FIELD OF INVENTION:
This invention relates to a recombinant rice catalase-B protein endowed with
light and temperature insensitivity in bacterial cytosol.
The present invention also relates to a process for the production of
recombinant rice catalase-B protein.
BACKGROUND OF THE INVENTION:
In general plant catalase (EC 1.11.1.6) is an enzyme that catalyzes the
decomposition of hydrogen peroxide to oxygen and water. Cells utilize
catalases, together with other cellular enzyme systems, to protect themselves
against the harmful effects of oxygen deleterious effect of hydrogen
peroxide (H2O2).
Attempts have been made in past to engineer this protein with various
change in its chemical characteristics to make use of the enzyme in medicine
and industries like pulp bleaching, textile bleaching, water treatment, as an
intermediate in the synthesis of other substances and other miscellaneous
uses.
The last 10 years have seen the application of DNA manipulation strategies
on catalase genes from various organisms. The first engineered catalase was
constructed in 1990 from Yeast peroxisome and open a new era of molecular
biology investigations in catalase.

Catalases are antioxidant proteins, found in all aerobic organisms and
functions as a detoxifier of reactive oxy-molecule; H2O2. The catalase in
photosynthesizing plants plays a central role not only in scavenging H2O2
under normal and stress full condition, but also indirectly participate in the
signal translocation cascade by maintaining the steady state level of the
peroxide in the cell.
The plant catalases so far examined are highly denaturable (i.e. a
conformational alteration resulting in the loss of biological activity) upon
exposure to high light intensity both under in vivo and in vitro conditions.
The enzyme is susceptible to deformity by its own substrate. None the less,
the protein also suffers from photoinactivation under moderate light
condition when remain exposed of low light intensities.
The plant catalase also suffers from denaturation at elevated temperature.
Generally the thermal denaturation increases in non-linear fashion with
increase in temperature. Therefore, the actual thermal denaturation of the
enzyme under elevated temperature is the sum of the product from
deactivation rate and the duration of incubation.

In addition to temperature, pH also affects enzyme kinetics and stability of
the enzyme. The pH may affect deactivation of the enzyme due to covalent
changes, such as the deamination of asparagine residues and non-covalent
changes such as the rearrangement of the protein chain. High pH, indicative
of a basic or alkaline environment, may also result in random cleavage of the
peptide. Beyond deamination and cleavage, pH has a substantial effect on
the protonation state of the amino acid side chains and the function of the
enzyme. Thus, enzymes display a range of pH within which they will
function adequately. In particular, commercially available catalases are
optimally active at a temperature range between 20-50°C.
Three general classes of catalases have been described in the literature: the
typical or monofunctional catalases; the catalase-peroxidases that have a
peroxidative activity as well as catalase activity; and the Mn-catalases or
pseudocatalases. Typical catalases, which have similar properties, have been
isolated from numerous animals, plants, and microorganisms. These
enzymes typically have four subunits of equal size with a combined
molecular mass of 225,000-270,000 kDa and characteristically have four
protoheme IX prosthetic groups per tetrameric molecule. These enzymes
also typically display a broad pH activity range from 4 to 10, are specifically
inhibited by 3-amino-l,2, 4-triazole.

Most of the plant catalase utilizes protoheme IX. Though the crystal
structure of the plant catalases are not yet available but they showed a high
degree of homology with other available catalases. Additionally, the plant
catalases typically have a sharp pH optimum, are inhibited by 3-amino-1, 2,
4-triazole, are also sensitive to hydrogen peroxide concentration, and are
readily reduced by dithionite. Sequence analysis of available plant catalase
enzymes has shown that they are related and on the basis of sequence
similarity, it strongly inhibited by cyanide and azide, bothe of which are
classic heme protein inhibitors.
The thermostable versions of catalases are mainly reported from various
bacterial sources. Many of these reported enzymes exhibited low thermal
stability at temperatures above 60°C; several were rapidly inactivated in the
presence of H2O2, and most of the enzymes had low activity and stability at
elevated temperature and pH, making them unsuitable for many applications.
But there is a huge void of find out such features from plant sources.
In contrast to the present invention available catalases from plant sources
exhibit little to no activity under conditions of elevated temperature and high
pH and also inactivated at high substrate concentration, suffer injury to low
temperature exposure.

OBJECTS OF THE INVENTION:
An object of this invention is to propose a recombinant rice catalase-B
protein endowed with light and temperature insensitivity in bacterial cytosol;
Another object of this invention is to propose a process for the production of
recombinant rice catalase-B protein and endowed with light and temperature
insensitivity in bacterial cytosol;
Further, object of this invention is to propose a recombinant rice catalase-B
protein which can function at high alkaline pH sustaining high pH exposure
without loosing its H2O2 scavenging activity;
Still further object of this invention is to propose a recombinant rice
catalase-B protein which is capable of functioning at high light intensity
under in vitro conditions without losing its activity.
Still another object of this invention is to propose a recombinant which has a
high affirmity for its substrate (H2O2).

BRIEF DESCRIPTION OF THE INVENTION:
According to this invention there is provided a genetically engineered rice
catalase-B from E.coli comprises antioxidant protein that can express in
M15 strain of E.coli bacterial cytoplasm when coexist with bacterial
chaperonin GroEL/ES wherein it is endowed with relatively higher
resistance to elevated temperature treatment and is capable of functioning at
high light intensity up to 2h under in vitro conditions without loss of any
activity.
In accordance with this invention there is provided a process for making a
genetically engineered rice catalase-B comprising :
developing rice catalase-B protein by mutagenesis of specific amino acids;
subjecting the said protein to the step of recombinant expression in E-coli.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
Fig 1. shows a 3-D In-silico modeling of rice CAT-B monomer unit .The six
substituted residue are follows V124, LI35, LI89, G205, H225;
Fig 2: shows schematic diagram of in vitro site-specific recombinant
polymerase chain reaction (PCR) technique. Down ward red arrows
indicate the sites of mutation.

Fig 3 A: shows the sequence of native (control) CAT-B is shown on the left
with sequences of the same region for the mutation L189W shown
on the right. Base changes required for replacement of Leul89
(CTC) in the wild type enzyme with the amino acid Trp (TGG) are
indicated below the autoradiogram and also see the square
brackets.
Fig 3B: shows the sequence of native (control) CAT-B is shown on the left
with sequences of the same region for the mutation H225T shown on
the right. Base changes required for replacement of His225 (CAC) in
the wild type enzyme with the amino acid Thr (ACC) are indicated
below the autoradiogram and also see the square brackets.
Fig 3C: shows the sequence of native (control) CAT-B is shown on the left
with sequences of the same region for the mutation K291M shown
on the right. Base changes required for replacement of Lys291
(AGG) in the wild type enzyme with the amino acid Met (ATG) are
indicated below the autoradiogram and also see the square brackets.
Figs 4A, B and C: Shows Confirmation of directional cloning (ATG-STOP)
of CatB in pQE30UA vector. Figure-4A shows the
EcoRV restricted digestion; mark the presence of 900 bp
insert. Figure-4B also suggests for directional cloning.
Lane 1 in Fig. 4B shows the native vector including CatB
(pQE30UA-CatB). Lane 2 of Fig. 4B represents the

EcoRV digestion pQE30UA-CatB construct. Lane 3 in
Fig. 4B denotes the CatB PCR product obtained using
CatB specific primer with pQE30UA-CatB as templates.
Figure-4C depicts the restriction digestion map of CatB
ORF (NEB Web-Cutter, V.2.0). The internal EcoRV
restriction site has been shown against arrow mark.
Figs 5A and B: Shows SDS-PAGE analysis showing the expression profile
of recombinant CAT-B protein in bacterial system. 5(A)
portrays the IPTG induced (2) CAT-B expression (marked as
arrow), in contrary to non-induced (1). M15 bacterial were
taken as control. The superiority of CAT-B expression in
bacterial system with 2 x YT as growth medium is examined
in figure-5B. Other notations in 'B' is same to that of 5 'A'
Fig 6: Shows 10% SDS-PAGE analysis of rice CAT-B (pQE30UA-CatB)
recombinant protein from periplasmic (PP) cytoplasmic (CY) and
inclusion body (IN) fractions under IPTG-uninduced (1) and IPTG-
induced (2) conditions. Mark the presence of IPTG-induced CAT-B
protein shown against arrow.
Fig 7: shows relative molecular weight of recombinant CAT-B. The
depicted figure is a negative impression of the 10% SDS-PAGE.

Fig 8: shows the products from in vitro site-specific recombinant
polymerase chain reaction (PCR). Lane 1 and 2 respectively
denotes the 1st PCR analysis with 5'F and internal mutated
antisense primer (A565mR) and 3'R and internal mutated sense
primers (A565mF). Lane 3 and 4 depicts the 2nd PCR with 5'F and
3'R using 1st PCR products as template. The 'M represents the
marker.
Fig 9: shows 10% SDS-PAGE analysis of native (pQE30UA-CatB) and
variant (L189W/H225T, L189W/H225T/K229M) rice CAT-B
recombinant protein from cytoplasmic (CY) and inclusion body (IN)
fractions under IPTG-uninduced and IPTG-induced condition. The
presented photograph is the negative picture of the CBB stain.
Figs 10A, B & C: Shows 10% SDS-PAGE analysis of recombinant rice
catalase protein (54-kDa) in cytoplasmic fraction of bacterial
extract both in absence (panel A, lane 1) and presence (panel A,
lane 2, 3 and 4) of GroEL/ES (56-kDa). Lane 3 and 4 in figure A
shows respectively the expression of mutated catalase 189W -225
T and 189 W - 225 T - 291 M in presence of GroEL/ES. The'
heterologous expression of recombinant rice catalase in bacterial
cytoplasm was identified by western blot analysis with antisera of
rice plant catalase (panel B) and with immunobloting with
histidine specific antibody (panel C). The lane numbers in panel B
and C are of similar condition as mentioned for panel A.

Figs 11 A, B & C: Shows (A) SDS-PAGE analysis of cytoplasmic (CY) and
inclusion (IN) body protein fraction of E.coli. The over
expressed recombinant protein in the inclusion body has
been indicated as star mark. (B) 10% SDS-PAGE
analysis native and variant CAT-B protein (gel photo is
the negative picture of the CBB staining). (C) Histogram
depicting the specific activity of control and variant
CAT-B protein,. The vertical bar represents the ± SD of
mean activity obtained at least from three independent
experiments.
Figs 12A & B: Shows dependence of enzyme velocity on H2O2
concentration. The open and closed circles respectively
represent the activity in control and variant catalases.
The inset in figure A is the larger version of the main
figure. Figure 34B depicts the Michaelis-Menten
kinetics drawn from the data obtained in figure A.
Fig 12C: shows in silico modeling of rice catalase-B, developed based on the
structural similarity and known crystal structural of CatF. The
specific mutation sites like H225T and L189W are marked in blue
and green respectively. The presence of heme moiety is shown in
red. The 'N' and 'C terminal ends of the protein have been shown
by alphabets N and C.

Figs 13A & B: Shows effect of light on native catalase-B (closed symbols
with solid line), and variant (open symbols with solid line)
recombinant rice catalase activity obtained from cytosolic
fractions (A) and in vitro renatured protein (B). 25 µg
protein in 1 ml of air saturated 50 mM Tris-HCl buffer (pH
7.5) was exposed to light (≈ 800 µE m-2 s-1) at 25 °C for
varied time duration having respective dark controls (closed
symbols with dotted line). The results of three independent
experiments have been depicted by different symbols.
Fig 14A & B: Shows Catalatic activity of native and variant recombinant
rice catalase-B purified from bacterial cytoplasm. The
comparison of the relative temperature sensitivity among the
constructs has been depicted subsequent to normalization of
their activities obtained at 25 °C incubation. Hydrogen
peroxide scavenging activity of native and variant catalase-B
recombinants, incubated at 10 °C for varied time period
under light (800 µE m" s' ) has been shown in figure 14 (B).
In both experiments, recombinant protein (25 µg/ml in 50
mM K-PO4 buffer, pH 7.5) was incubated at the desired
temperature in a temperature controlled cuvette and exposed
to white light of ≈ 800 µE m-2 s-1 . Following the temperature
treatment, the samples were acclimatized to room
temperature (25 °C) before activity measurements. The

vertical bars represent the ± SD of four independent
experiments.
DETAILED DESCRIPTION OF THE INVENTION:
The invention relates to a light insensitive rice catalase protein express in
bacterial system. Nucleic acid sequences encoding the catalase, with site
directed mutagenesis of the specific amino acid residues to impart light and
temperature insensitivity that catalyzing the conversion of hydrogen
peroxide to water and oxygen.
The invention relates to a catalase having an activity after incubating for few
hours at a particular temperature and a pH of about 10.0 and that
demonstrate substantially no substrate inhibition at hydrogen peroxide
concentrations up to about 20 mM. An exemplary catalase was obtained
from genetically modified rice plant catalase B.
In a further exemplary embodiment, the invention also relates to an isolated
light and temperature rice catalase produced as an over expressed protein in
bacterial system in presence of GroEL/ES. Preparing a cell lysate from the
microorganism; identifying a catalase activity in the cell lysate; purifying the
catalase activity from the cell lysate; demonstrating the absence of
substantial substrate inhibition of the catalase activity at a H2O2
concentration between about 20mM; and have appreciable H2O2 scavenge
activity in temperature insensitive manner.

The invention also reports IMPORTANTLY the expression of active soluble
plant catalase in the cytosol of E.coli with the presence of chaperone
(GroEL/ES). In an additional exemplary embodiment, the invention relates
to a method of converting hydrogen peroxide to oxygen and water under
conditions of both high/low temperature and alkaline pH.
The invention further relates to an isolated nucleic acid comprising a nucleic
acid sequence with specific mutation encoding a polypeptide having the
sequence for catalase enzyme endowed with light and temperature
insensitiveness.
This invented nucleotide sequence can cloned in to a suitable expression
vector; for expression of light and temperature insensitive catalase protein in
a specific host.
The invention also relates to functional fragments of the catalase. The
catalase of the invention includes fragments of the catalase wherein catalase
activity is retained.

The invention also relates to a host cell containing a nucleic acid encoding a
light and temperature tolerant catalase. For example, the host cell may be
used to express the catalase and may be used as a means of producing the
catalase.
The recombinant catalase-B protein producing genetic material along its host
has been deposited to Microbial Type Culture Collection and Gene Bank
(MTCC), Institute of Microbial Technology, Sector-39A, Chandigarh-
160036, INIDA, and the Accession number allotted is MTCC 5372.
The invention further relates to an isolated nucleic acid comprising a nucleic
acid sequence with specific mutation encoding a polypeptide having the
sequence for catalase enzyme endowed with light and temperature
insensitiveness.
This invented nucleotide sequence can cloned in to a suitable expression
vector; for expression of light and temperature insensitive catalase protein in
a specific host.
The invention also relates to functional fragments of the catalase. The
catalase of the invention includes fragments of the catalase wherein catalase
activity is retained.

The invention also relates to a host cell containing a nucleic acid encoding a
light and temperature tolerant catalase. For example, the host cell may be
used to express the catalase and may be used as a means of producing the
catalase.
A genetically engineered rice catalase-B from E.coli comprises antioxidant
protein that can express in M15 strain of E.coli bacterial cytoplasm when
coexist with bacterial chaperonin GroEL/ES. This form of rice catalase-B
has been invented for the first time and no literature information exists for
the used host (E. coli).
The recombinant rice catalase-B from E.coli endowed with relatively higher
resistance to elevated temperature treatment than the normal rice catalase-B.
The genetically engineered rice catalase-B endowed with characteristics of
low temperature resistance of about 10°C for nearly 2 hours without any loss
of activity than the normal rice catalase-B. The genetically engineered rice
catalase-B that is capable of functioning at high light intensity up to 2h
under in vitro conditions without loss of any activity. The genetic engineered
rice catalase-B can be function at high alkaline pH substaining high pH
exposure up to 10 with out losing its' H2O2 scavenging activity. The genetic
engineered rice catalase-B does not show substrate mediated inactivation
upto 20 mM H2O2 concentration. The genetically engineered rice catalase-B
with high affinity for its substrate (H2O2) due its low Km (4 mM) than
normal rice catalase B (11 mM). The genetically engineered rice catalase B

with high H2O2 scavenging velocity (Km/Vmax) that is nearly 3 fold high
than the normal rice catalase-B. The genetic engineered rice catalase-B with
higher catalatic activity at any given pH range (6-10) than the typical rice
plant catalase-B. The genetic engineered rice catalase-B with high catalytic
activity at low H2O2 concentration compared to normal rice catalase-B. The
genetically engineered rice catalase-b with a high turn over rate than normal
rice catalase-B at low substrate concentration. The genetic engineered rice
catalase-B expressed in E.coli consists of four identical subunit of
approximately 54kDa for a total molecular mass of 240 kDa. The genetically
engineered rice catalase-B DNA, refers to a fragment of DNA that has been
mutated at specific nucleotides positions to obtain a genetically engineered
rice catalase-B protein having light and temperature resistance
characteristics.
Recombinant expression of CAT-B
Cell lines and plasmids
M15 E.coli strain, K12 derivative was used for the expression of
various plasmids. M15 E.coli strains, containing multiple copies pREP4
plasmids was maintained in presence of kanamycin. pREP4 plasmid carries
the lad gene encoding the lac repressor thus facilitating the expression of
various foreign genes. Rice catalase gene was cloned in to cloning vector,
pQE30UA (Qiagen) with a ColE1 origin of replication. The pREP4 plasmids
are compatible with all plasmid carrying the ColEl origin of replication and
pQE30UA vector carried T5 promoter that enables the expression from

translation start site with the cloned insert and N-terminal fusion. pKY206,
derived from pACYC184, carried the GroEL/ES gene and can co-exist in the
same cells with the sub cloned plasmids (Mizobata et al. 1992).
Construct preparation for protein expression
The cDNA for the rice CatB (NCBI accession no DO078758), was
cloned into pGEM T easy vector. In order to clone into expression vector,
CatB was PCR amplified from pGEMT-CatB construct using CatB gene
specific primer (Table 1 )The amplified DNA fragment was gel purified and
was ligated into the pQE30UA. The construct pQE30UA-CatB was used to
transform the competent E.coli M15 cell lines following ampicillin selection.
Several clones were harvested; positive clones were selected through colony
lysis PCR and restriction digestion.
Table-1: Name and sequences of oligos used for various PCR analyses. 'F' and 'R' stands for
forward and reverse primers used in the experiments. Specific nucleotide mutational sites have
been shown as bold face lower case letters.


Site directed mutagenesis
Recently Engel et al.2006 have shown the recombinant catalase of
H.alpina is completely resistance to the photoinactivation. A multiple
sequence alignment of H. alpina and rice CatB cDNA showed a high
homology between them and all other residues that are responsible for heme
binding and catalytic activity (Fita and Rossmann 1985; Zamocky and
Koller 1999; Nicholls et al. 2001) are strictly conserved. Only six amino acid
substitutions can regard as exceptional. It is fairly obvious that these 6
residues can be expected to contribute to the catalase extraordinary
prosperity of light insensitiveness.
Homology modeling (Sekhar et al. 2006) of rice CAT-B has shown
that the substituted amino acid that are specific for light insensitive catalase
are localized in a restricted area of the molecule. Marked the presence of
those residues (Figure-1).
All the six unusual amino acid substitutions occur with in the central
antiparallel P-barrel domain comprising strands β1- β8 (Zamocky and Koller
1999; Nicholls et al.2001). Among the six for the present study the most
exceptional acid substitution of the light stable catalase -Thr225 on the β6,
Met291 between strands β7 and β8 and Trp189 on the helix α5- all are
localized at or near the surface of the molecule at entrance of the minor
lateral channel leading to the distal side of the heme. Current study picked
up the above mentioned three amino acid residues to rationalize their role in
conferring light insensitivity. Two Mutant constructs was used in this report

one was with two amino acids substitutions L189W/H225T, and another
with three L189W/H225T/K291M.
The double variant L189W/H225T and triple variant
L189W/H225T/K291M of CatB were synthesized using an in vitro site
specific recombinant polymerase chain reaction (PCR) technique (Higuchi
1990). Two separate PCR reactions were performed, using the sense or
antisense sequence of the required mutated region and the 5' and 3' flanking
region primer (Table 1). The first PCR was done with 5'F, and antisense
primers and 3'R and sense primers respectively. The PCR products were gel
purified and used as template in a second PCR reaction with 5'F and 3'R
primer sets. The 5'F primer includes EcoRV at the 5' end for cloning
(Figure-2). The final PCR product was ligated in to the EcoRV site of
pQE30UA. All the PCR reactions were performed using Tli polymerase
(Promega) and the mutated clones were manually sequenced to confirm the
specific mutation sites (Figure- 3 A, B, and C,).
Recombinant expression of Catalase protein
Transformed E.coli (M15) cells were cultured overnight at 37 °C in 5
ml 2X YT medium containing ampicillin or ampicillin + kanamycin. One ml
of over night culture was then inoculated in to 100 ml of 2 x YT containing
the appropriate antibiotics; Cultures were maintained at 37 °C with shaking
until cell density reached to an A600 = 0.6. Cells were then induced with 1
mM isopropyl ß-thiogalactopyranoside (IPTG) and allowed to grow for
additional 3-h under same conditions. Bacterial cells were harvested by

centrifugation at 12, 000 x g, 4 °C for 20 min and the pellets were re-
suspended in 1 ml of 30 mM Tris buffer (pH 8.0) containing 1 mM EDTA
(pH 8.0), 20% sucrose. The suspensions were put on ice for 10 min, and
centrifuged at 12,000 x g for 10 min (supernatant is periplasmic fraction),
and the resulting pellets was re-suspended in 5 ml 50 mM Tris buffer (pH
8.0) containing 2 mM EDTA, 0.1mg/ml lysozyme, 1% triton X-100 and
1mM ATP. The suspension were incubated at 30 °C for 20 min and
sonicated in cold for minimum of 10 cycles of 40 W. The clear lysate were
treated with 100 µg/ml DNasel and RNase for 10 min at 4 °C and then
centrifuged at 12, 0000 x g for 15 min at 4 0C. The supernatant constitutes
the cytoplasmic fraction. The pellet containg the bacterial inclusion bodies
were solubilized for over night with minimum volume of solubilized (0.1 M
Tris-HCl (pH 8.0), 2 mM EDTA, 0.3 M DTE and 6 M urea). The insoluble
materials were separated under high centrifugal force of 25, 000-x g for 30
min. The proteins of different fractions of E. coli were kept frozen at -20 °C
until use (Bai et al.2003)
Inclusion body protein solubilization and renaturation of solubilized
protein
Inclusion body pellet was suspended in 50 mM Tris-HCl buffer (pH 8.0)
containing 6M GdnHCl at 4 °C. The un-solubilized fraction was removed by
centrifugation at 25, 000 x g for 30 min. Protein concentration in the
supernatant was determined following Bradford (1976). Various
combinations of salts, redox active compounds in Tris-HCl buffer system for

renaturation of solubilized CAT-B protein from inclusion body. The
composition having 0.99 M GdnHCl, 0.8 M L-arginine, 0.05 M Tris-HCl
(pH 8.2), 0.02 M NaCl, 0.8 mM KC1, 1 mM EDTA, 2 mM GSH, and 0.4
mM GSSG was found to be most ideal in obtaining the functional
renaturation of control and variant forms of CAT-B. Measured amount of
denaturated (0.025-0.050 mg) protein was incubated with the above reaction
medium in a total volume of 1 ml at 4 °C for 24-h. Low temperature
incubated samples were further exposed to 30 °C for 2-h.
Partial purification of renaturated CAT-B
Two (2) ml of renaturated protein (25 µg) solution was loaded onto a
Superdex-200 gel filtration column (15cm x 1 cm) that was pre-equilibrated
with 10 mM Tris-HCl buffer (pH 8.5). The protein was eluted in 1.5 ml
fraction sizes under 2 ml min-1 flow rate. Fractions showing catalase
activity were pooled together and dialyzed extensively with 10 mM Tris-
HCl buffer (pH 8.5) for nearly 48-h with 6 changes of buffer solution. The
dialyzed protein was concentrated on a bed of solid PEG (8000 MW) at 4
°C. The concentrated protein was centrifuged at 25, 000 x g for 30 min and
preserved at 4 °C until use. The protein stored at 4 °C could retain its'
activity for nearly 15 days but a loss of 25 to 30 % activity was marked on
long term storage (over 30 to 35 days). The purity was checked by 10%
SDS-PAGE stained in CBB.

Western analysis
Protein blotting to PVDF membrane
Recombinant catalase was expressed as a fusion protein having (His)6
at its N-terminal end. The protein from cytoplasmic fraction was semi-
purified using Ni+-affinity column chromatography (Pierce kit). Semi-
purified catalase was desalted (Pierce desalting column) and assayed for
catalase activity and also immuno-reacted with rice catalase anti-sera and
histidine specific antibody by Western blotting following the
chemiluminescence's protocol as described in Pierce's super signal kit.
Proteins separated by SDS-PAGE (Laemmli 1970) were transferred onto the
activated immuno blot PVDF membranes (Sigma) with semi dry blotting
apparatus according to company manual. The membrane was first incubated
in the blocking buffer, contains 5% (w/v) fat free milk powder in TTBS
(100mM Tris (pH 7.4), 150mM NaCl and 0.2% (v/v) Twen20) for 2 h in
room temperature. Subsequently, the solution was discarded and the fresh
blocking solution containing the primary antibody was added and incubated
for 1 h under the same conditions. After washing 5 times for 10 min with
TTBS an aliquot of blocking solution containing the secondary antibody
labeled with HRP (1:5000 dilutions) was added and incubated for 1 h. The
membrane was rinsed 5 times with TTBS before protein diction.

Assay of catalase activity
Catalase activity was assayed following the method as described by
Aebi (1983). The reaction mixture contained 20 µg equivalent proteins in 50
mM phosphate buffer (pH 7.0) and 10 mM H2O2. The decrease in
absorbance at 240 nm (due the consumption of H2O2.) was recorded using a
UV-visible spectrophotometer (Varian, Carry 100 Biol). The activity of
catalase was calculated using the extinct co-efficient of 40 M"1 cm"1 for the
peroxide at 240 nm and expressed as mol quantity of H2O2.consumed mg-1
protein h-1
In order to identify if the recombinant catalase is plant specific,
inhibitory affect of 10 mM AT was examined. The light responsiveness of
normal and mutated catalase was studied by exposing the protein to white
light (800 µE m-2 s-1) with proper dark control in a temperature controlled
(25 °C) water-jacketed vial for specific time period (see results). The
temperature susceptibility was evaluated by incubating the protein at varied
temperatures in dark. Other details of reaction kinetics have been included in
legends to respective figures. Enzyme velocity was determined from initial
10 s of the reaction. The sensitivity of enzyme activity to increasing
concentration of H2O2 was measured by varying the substrate concentration
in the range of 1-50 mM.
The O2*- sensitivity of the recombinant catalase protein from both
control and mutant form were made in twenty-five µg of protein incubated
with or without 1unit of xanthine oxidase for 1h in presence of 15 µM
acetaldehyde, 0.1 mM Na-EDTA in 50 mM K-PO4 buffer (pH 7.5) for 2 h at

25 °C in dark. The catalatic reaction was measured at 240 nm for a period of
300 s in presence of 10 mM H2O2.
Cloning of CatB in pQE30UA vector
Complete cds of rice CatB was cloned in "Uracil" overhang
pQE30UA vector. Restriction digestions with EcoRV validate the directional
cloning of CatB in expression vector (pQE30UA). Since both vector and
CatB (internal site 888bp form ATG site) is having the EcoRV sites, the
EcoRV restriction enzyme was preferred as a selector enzyme for orientation
confirmation of cloned product (Figure- 4).
Over expression of the rice Catalase-B in E.coli
E.coli is used extensively for expression and overproduction of both
prokaryotic and eukaryotic recombinant proteins, as it requires very simple
growth condition (Baneyx 1999). An extremely high level of expression of
rice CAT-B was achieved with the induction of IPTG (lmM) in cells
harboring pQE30UA-CatB plasmid (Figure 5A), both in 2xYT and super
broth medium. Induction profile suggests (Figure- 5B) the superiority of
2xYT than super broth medium. Thus 2xYT was selected for rest of the
protein expression study. The over expressed protein was, however, found
to restrict themselves inside the bacterial inclusion body fraction (Figure -6).
The apparent molecular mass of the inclusion body protein was found to be
nearly 54 kDa. (Figure-7).

The SDS-PAGE gel purified protein band corresponding to CAT-B was
further analyzed by in-gel trypsin digestion. The trypsin digestion and its
subsequent MALDI-TOF-MS (Matrix assisted Laser Desorption Ionization -
Time of flight) analysis also confirmed that the recombinantly
expressed protein in E.coli is rice catalase B.
Generation of CAT-B mutants and their recombinant expression
The CAT-B mutants (L189W/H225T, L189W/H225T/K229M)
were generated by in vitro site directed mutagenesis PCR techniques
(Figure-8). The native and mutated CafB gene was further expressed
in E.coli system. As found for native protein, the mutated proteins
also showed appreciable expression in bacteria but the expressed
protein was remain confined to inclusion body fraction (Figure-9).
The specific mutational sites were validated by manual sequencing (Figure-3
A, B and C).
Recombinant expression of rice Catalase-B in bacterial cytoplasm Effect of co-
expression of Cat-B with GroEL/ES
Heterologous expression of functional plant catalase protein in E.coli
expression system has so far unsuccessful (Zamocky and Koller 1999; Engel
et al. 2006). However, an attempt was made to express a rice catalase-B
encoded protein in E.coli with co-expression of chaperonin, the GroEL/ES.
IPTG induction to transformants harboring pQE30UA-CatB showed that

presence of GroEL/ES can stimulate the synthesis of recombinant rice
catalase-B protein in soluble form (Figure-10A, lane 2 and 3) but to a
reduce quantity. These results suggest that the over expression of GroEL/ES
in E. coli expression system could help in refolding the rice catalase protein
that otherwise remained in inclusion body in inactive form in absence of the
chaperonin. The presence of rice recombinant catalase protein in soluble
form in bacterial cytoplasmic fraction was confirmed with western blot
analysis using rice catalase anti-rabbit anti-sera (Figure-10B, lane 2 and 3)
and with monoclonal anti-mouse His-antibody (Figure-10C, lane 2 and 3).
The antibody reactivity while clearly discernible for native and double
mutated forms (L189W/H225T) of recombinant rice CAT-B, it however,
failed to react with recombinant protein produced as triple mutated
(L189W/H225T/K291M) form (Figure-10 B and C). In addition, the triple
mutated purified protein was also devoid of catalase activity. Therefore, the
triple mutated form of recombinant catalase was not considered in further
experiments. As reported previously (Gupta et al. 2006), co-expression of
GroEL/ES caused a slower growth rate bacteria compared to expression of
only CAT-B
Biochemical characterization of recombinant rice catalase-B expressed
in E. coli
Renaturation of protein from inclusion body

The recombinant proteins of native and variant form, housed in inclusion
body of the bacteria (Figure-11A) were renaturated, purified through gel
filtration and its purity was monitored by SDS-PAGE analysis (Figure-
11B). The renaturated proteins of both control(native) and variant forms of
recombinant CAT-B showed the presence of an intense CBB stained protein
band having about 54-kDa molecular mass (Figure-11B ) suggesting that
the protein is largely purified one and is comparable to the monomeric
molecular mass of rice catalase. The H2O2 scavenging activity, expressed per
mg protein basis was noticed to be different in native and mutated forms of
catalase (Figure-11C). A nearly 2 fold high catalatic activity was noticed
with the variant form compared to the native one.
Inhibitory effects of aminotriazole
Aminotriazole inhibits plant or animal catalase activity (Margoliash et al.
1960) but not to bacterial one (Switala and Loewen 2002). The inhibitor
disrupts the catalatic function of the enzyme by its binding to compound-I.
This compound has been implicated as a specific inhibitor of catalase in
plant and animal systems in presence of H2O2 that is utilized for the
formation of compound-I. (Margoliash et al. 1960). The inhibitory effect of
AT was examined both in native and mutated recombinant rice catalase-B.
The both native and mutated rice catalase activity was inhibited in an AT
concentration dependent manner having about 50% inhibition at 10 mM of
the compound. As expected, the bacterial catalase activity, assayed from the
crude extract remained completely insensitive to the inhibitor (Table-2).

Inhibitor studies additionally suggest that the purified proteins are the
recombinant rice catalase protein.
Table-2: Aminotriazole (AT) concentration dependent inhibition of
bacterial (non-induced), purified native (pQE30UA-CatB) and variant
(L189W/H225T) recombinant rice catalase-B obtained from inclusion body
fractions. ± values represent the SD of mean observation obtained from three
independent batches of experiments. Data has been expressed as µmol of
H2O2 consumed mg protein-1 min-1.


Steady state kinetic comparison between native and variant CAT-B
The H2O2 scavenging ability of both the forms of catalases was
examined under varied concentration of H2O2 (Figure -12 A,). The enzyme
activity showed an initial linear increase in velocity with increasing
concentration of substrate but stopped abruptly and at higher H2O2
concentration (Figure- 12A) of near about 58-60 mM the velocity start
declining, it was noticed that the concentration of H2O2 required to initiate
the inactivation of activity in native and variant forms of CAT-B was
slightly different; the variant requiring a relatively higher concentration (2 to
4 mM more) of the substrate than native one for its' inactivation (Figure-
12A inset). These findings suggest that the site-specific mutation of amino
acids not only results in higher scavenging ability of H2O2 but also endowed
with characteristic to resist the substrate-mediated inactivation of its activity.
The substrate concentration required for achieving half maximal
velocity in an enzyme-substrate reaction determines its' catalatic efficiency
which is represented as Michaelis constant or Km. However, the classical
terms of Vmax and Km cannot be directly applied for catalase reaction because
of non-existence of typical substrate dependent saturation kinetics. An
apparent Km value for catalase could be determined, using the initial velocity
of the enzyme substrate reaction by Lineweaver-Burk plot analysis a more
theoretical interpretation than reality. Similar analysis of data from figure
showed that these two forms of recombinant catalases differ significantly in
their apparent Km value. The apparent Km for native-form was calculated to
be ≈ 11 ± 2 mM, while a significant lower Km (≈4 ± 1.2 mM) was evident

with the variant from of rice CAT-B (Figure-12 B, see the inset). The
theoretically calculated Vmax of both the forms of catalase did not show much
variation. The H2O2 scavenging velocity (Km/Wmax) of the variant form was
nearly 3 fold high than the control, which suggests that the variant enzyme
has a high affinity for its substrate.
Our further analysis indicated that the variant form of rice CAT-B is
relatively more efficient in scavenging H2O2 at low concentration of
substrate having high enzyme turnover rate as compared to control form. A
comparative statement of the velocity and the enzyme turnover rate has been
depicted in table-3, page 78.
Table-3: Relative enzyme velocities and the turn over rate of native and
variant recombinant rice catalase-B at H2O2 concentrations yielding ½ Vmax ;
4 mM for variant and 11 mM for native. The bracketed numbers refers to the
turn over rate of the enzyme, expressed as mole of substrate consumed per
mole of enzyme min-1. The mean value of enzyme activity from three
independent experiments was considered for the determination of turn over
rate. The ± represent the SD of enzyme velocity determined from three
independent experiments.


An in silico three dimensional model of CAT-B based on the crystal
structure of CatF (PDB code lm7s) was developed following protocol as
mentioned by Sekhar et al. (2006) and the putative position of the substituted
amino acids were marked (Figure-12). The mutational effect was also
reflected in the H2O2 scavenging ability of the enzyme; the variant having a
relatively higher efficiency than the native one. Passage of substrate H2O2
through one or more channels to the deeply buried active site (heme) is
essential for catalase activity (Switala and Loewen 2002). The observed
differences in the velocity for scavenging H2O2 among the native and variant
forms of recombinant catalase in the present investigation may have been
arisen for differential rate of accessibility of the substrate to the site of
dismutation. The substitute amino acids in variant form, Trp-189 and Thr-
225 (Figure-12) are placed on the helix α-5 and β-6 strand respectively at
the entrance of minor lateral channel leading to the distal side of the central
heme (Engel et al. 2006). Therefore, the mutational effect causing an

enlargement of the entrance of lateral channel may have resulted in a marked
increase of specific activity of the mutated protein by increasing the
accessibility of the substrate to the site of dismutation.
A mutation causing an enlargement of the lateral channel in E.coli catalase
HPII (catalase-peroxidase) has been shown to increase its specific activity
due to high rate of substrate accessibility to the site of dismutation (Sevinc et
al. 1999). A similar conclusion thus may be drawn for mutational mediated
increase in specific activity of variant rice CAT-B in this investigation
(Mondal et al. 2008).
Kinetics of catalatic activity of recombinant rice catalase-B protein:
Effect of environmental stresses
Sensitivity of engineered catalase-B to light and temperature
Replacement of amino acid lucine at 189 and histidine at 225 positions in
normal rice catalase with tryptophan and threonine respectively, drastically
altered the enzyme sensitivity to light and temperature. When exposed to
white light of nearly 800 µE m-2 s-1 for 120 min, the native recombinant
catalase become maximally inactivated by nearly 50 % under a period of 2 -
h exposure. The variant catalase-B, under identical light treatment was not
inactivated by light (Figure-13 A and B). These observations suggest that the
specific site directed mutation of the two amino acids, specifically induce

light insensitivity property to some degree to rice catalase. However, the role
of these amino acids in resistance to light inactivation remains to be probed.
The striking difference observed between the normal and the mutated
catalase was their differential response to elevated temperature. The
enzymatic activity of both normal and mutated rice catalase-B protein was
sensitive to high temperature inactivation (Figure-14). However, the
temperature-induced inactivation of enzyme activity was comparatively high
in normal recombinant catalase as compared to the mutated one.
Normalization of enzyme activity of both the samples at 25 °C showed that
the mutated catalase retain nearly two fold higher activity at elevated
temperatures than the normal one. These observations indicate that
alternation in two specific amino acids at specific positions of the protein
that could confer light insensitivity may also be associated for inducing
some degree of resistant towards high temperature mediated deactivation of
the enzyme activity. However, there was the difference between the native
and variant in term of their sensitivity to elevated temperature. Whether this
apparent correlation between heat and light resistance is a result of a
common protective mechanism or a coincidence will require further study.
Invention claim functioning of a successful genetically engineered
CAT-B, endowed with characteristic of high H2O2 scavenging activity with
relatively low Km (high affinity for substrate) than the native rice CAT-B. A
specific mutation of these two amino acid residues in rice CAT-B has also
developed resistance towards photoinactivation of the protein irrespective of

temperature. Whether this apparent correlation between increase velocity
and resistance to light impairment is result of a common mechanism or a
coincidence will required further study.
The engineered catalase with light and temperature insensitiveness
and endowed with characteristics of low Km can be explored further in
transgenic research to evaluate the role of catalase in sustaining oxidative
stress in plants. The variant form of rice CAT-B with enhanced affinity
toward H2O2 and having appreciable higher scavenging activity may be of
value physiologically and deserves further biochemical and structural
characterization to decipher the proposed mechanistic detail in the line of
affective accessibility of substrate to the site of active reaction center of the
catalase.
EXAMPLES:
A method of converting hydrogen peroxide to oxygen
Process: adding a sample containing hydrogen peroxide to a catalase;
incubating the catalase with the hydrogen peroxide solution at a high
temperature and at an alkaline pH; and converting a desired amount of the
hydrogen peroxide to oxygen and water. The term "high temperature"
includes temperatures about 70°C and low temperature is as much as 10°C.

An alkaline pH includes pH values between about 8 and about 10 or any
range between about 8 and about 10, for example, between about 8.5 and
about 9.5.

We Claim:
1. A genetically engineered rice catalase-B from E.coli comprises
antioxidant protein that can express in M15 strain of E.coli
bacterial cytoplasm when coexist with bacterial chaperonin
GroEL/ES wherein it is endowed with relatively higher resistance
to elevated temperature treatment and is capable of functioning at
high light intensity up to 2h under in vitro conditions without loss
of any activity.
2. The genetically engineered rice catalase-B as claimed in claim 1 is
endowed with characteristics of low temperature resistance of
about 10°C for nearly 2 hours without any loss of activity.
3. The genetically engineered rice catalase-B as claimed in claim 1,
wherein said catalase-B can function at high alkaline pH sustaining
high pH exposure up to 10 with out losing its' H2O2 scavenging
activity.
4. The genetically engineered rice catalase-B as claimed in claim 1
wherein said catalase has high affinity for its substrate (H2O2) due
to its low Km (4 mM) and has high scavenging velocity (Km/V max).

5. The genetically engineered rice catalase-B as claimed in claim 1
wherein said catalase has high catalytic activity at any given pH
range 6-10.
6. The genetically engineered rice catalase-B as claimed in claim 1
comprises of four identical subunit of approximately 54kDa for a
total molecular mass of 240 kDa.
7. The genetically engineered rice catalase-B as claimed in claim 1
comprises of fragment of DNA that has been mutated at specific
nucleotides positions to obtain a genetically engineered rice
catalase-B protein having light and temperature resistance
characteristics.
8. A process for making a genetically engineered rice catalase-B
comprising:
developing rice catalase-B protein by mutagenesis of specific amino
acids;
subjecting the said protein to the step of recombinant expression in E-
coli.
9. The method as claimed in claim 8, wherein the said amino acids
are selected from Leu-189 to Trp-189 and His-225 to Thr-225.

10. The method as claimed in claim 8, wherein the said E-coli used is M15
E.coli strain, K12 derivative.

A genetically engineered rice catalase-B from E.coli comprises antioxidant protein that can express in M15 strain of E.coli bacterial cytoplasm when coexist with bacterial chaperonin GroEL/ES wherein it is endowed with
relatively higher resistance to elevated temperature treatment and is capable
of functioning at high light intensity up to 2h under in vitro conditions without loss of any activity.