Journal of Environmental Biotechnology
Vol. 8, No. 1, 35–41, 2008

Original paper (regular paper)

Purification and Molecular Cloning of Catalase from Rhizobium
radiobacter Strain 2-1
S.M. TANVIR HOSSAIN1, MAMI NAKAYAMA2, HIROO UCHIYAMA1 and TOSHIAKI NAKAJIMA-KAMBE*1
1

Graduate School of Life and Environmental Science, University of Tsukuba, 1–1–1,
Tennoudai, Tsukuba, Ibaraki 305–8572, Japan
2
S.G. Laboratory Co., 2–35 Ohorikoen, Chuo-ku, Fukuoka 810–0051, Japan* TEL/FAX: +81–29–853–4619* E-mail: toshi@sakura.cc.tsukuba.ac.jp(Received; 1 December, 2007/Accepted; 20 December, 2007)

A heat-stable catalase from Rhizobium radiobacter strain 2-1 was purified. The purified catalase showed an extremely
high specific activity of 764,500 units/mg and displayed stability in a broad pH range of 5–11 and thermostability up to
60°C. Its Km was 60.06 mM and its Vmax was 10.17 mM/min. The enzyme was considered to be composed of four identicalsubunits with a molecular weight 56 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtrationof 230 kDa. The gene encoding the catalase was sequenced by using touchdown and inverse PCR. The deduced amino acidsequence of the catalase from strain 2-1 showed 83% homology to that of the putative catalase Sphingomonas wittichii.
The proximal active site and the proximal heme ligand were tentatively identified. The combined results of the gene sequence
analysis and biochemical characterization proved that the catalase from strain 2-1 is a typical monofunctional heme catalase.
Key words: catalase, Rhizobium radiobacter

1. Introduction
Hydrogen peroxide is widely used for chemical treatment
in various industries particularly in the textile, paper, pulp,
food and dairy industries as a more environmentally-friendly alternative to chlorine that forms toxic and carcinogenic
chemicals called dioxins as by-products7,9,33). However, therapid formation of reactive oxygen species (ROS) fromhydrogen peroxide, including hydroxyl radicals is also a
threat6,10,25). In light of the growing concern over environmental problems, hydrogen peroxide should be decomposed
before disposal.
Currently, hydrogen peroxide is either diluted with large
quantities of water, which is expensive and wasteful, or
treated with agents such as sodium bisulphite or hydrosulphite, which leaves salt in the water5). Alternatively, the application of catalases has been suggested. These enzymes play
an important role in reducing the formation of the highly
reactive hydroxyl radical that arises from H2O2 degradation
via the Fenton reaction13). Catalases are assigned to threephylogenetically distinct groups: two groups comprise heme
catalases and one group comprises nonheme catalases8,22).
Generally, a few thermostable versions of a monofunctional
catalase32), catalase peroxidases4,12,32,34) and Mn catalases2,17)
have been described. True catalases are usually homo
tetramers, with a subunit size of approximately 60 kDa.
Each subunit contains one heme group20,21).

Since the hydrogen peroxide bleaching step occurs atelevated temperatures and pH (>60°C and pH 9), commercially available catalases that are optimally active at 20–50°C
and at neutral pH. The pretreatment was recurred thetemperature and pH prior to their use23,27). From this, thermostable catalases hold a great potential for application in
the various diﬀerent industries due to the increasing trend
to develop environmentally friendly technologies.
The availability of catalase that can function at hightemperature and pH would be attractive for the above
mentioned applications. However, many of the reported
catalases exhibit low thermal stability, several are rapidly
inactivated in the presence of hydrogen peroxide, and mostshow low activity and stability at elevated temperature andpH, making them unsuitable for industrial applications30).
Nakayama et al. isolated strain 2-1 that can produce
high-activity catalase from industrial waste. From phylogenetic analysis, strain 2-1 was identified as Rhizobium
radiobacter, formally Agrobacterium tumefaciens35,36) (Nakayama et al., unpublished data). In this study, we describe
the purification of catalase from Rhizobium radiobacterstrain 2-1. The catalase showed extremely high activity andexceptional stability at elevated temperatures and pHs compared with many other reported catalases. Furthermore, wehave determined its N-terminal amino acid sequence andcloned the catalase encoding gene. The alignment result ofcloned gene showed no homology with those of otherHOSSAIN et al.

36

known catalases.
2. Materials and Methods
2.1. Bacterial strain cultivation
The Rhizobium radiobacter strain 2-1 that was isolated
as an excellent catalase producer and described previously,
was used throughout this study. The medium composition
for catalase production (PYS medium) consists of peptone,
10 g; yeast extract, 5 g; and NaCl, 5 g in 1 l of deionizedwater. Strain 2-1 was cultivated aerobically up to the earlystationary growth phase at 30°C with reciprocal shaking at
200 rpm. The cells were harvested by centrifugation at
8,000×g for 10 min at 4°C. The cells (4 g wet weight) weresuspended in 4 ml of 50 mM potassium phosphate buﬀer(pH 7.0).
2.2. Catalase extraction
The cells (4 g wet weight) were suspended in 4 ml of
50 mM potassium phosphate buﬀer (pH 7.0). Ethanol wasadded at a final concentration of 1% as a catalase stabilizer.
Then, the cells were disrupted trice using a French Pressure
Cell (SIM Aminco, NY, USA) at a pressure of 12,500 psi.
The treated cell suspension was centrifuged with reciprocal
shaking at 25,000 rpm for 60 min to remove cell debris. The
crude enzyme solution was incubated overnight at 50°C andthen chilled at 4°C overnight. The resulting precipitate(probably composed of phospholipids and denatured proteins) was removed by centrifugation at 15,000 rpm for
10 min and the supernatant was used for column chromatography.
2.3. Ion-exchange chromatography
The crude extract was applied to a RESOURCE Q column(6.0 ml bed volume, GE Healthcare UK Ltd., England)
equilibrated with 20 mM potassium phosphate buffer(pH 7.0) at a flow rate of 5.0 ml/min. After washing thecolumn with the same buﬀer, the enzyme was eluted bylinearly increasing the ionic strength of NaCl over the range
of 0–0.5 M in the same buﬀer. Catalase activity was detected
at concentrations of 0.2–0.3 M NaCl. The active fractions
were collected and concentrated by ammonium sulfate precipitation.
2.4. Gel permeation chromatography
A superose-12 (GE Healthcare UK Ltd., England) column(1.6×50 cm) was equilibrated with 20 mM potassium phosphate buﬀer (pH 7.0) containing 0.2 M NaCl at a flow rateof 0.6 ml/min. The concentrated solution showing catalase
activity prepared in the previous step was then applied tothe column. Fractions showing catalase activity were pooled
as purified catalase.
2.5. Activity assays
Catalase activity was measured spectrophotometrically bymonitoring the decrease in absorbance at 240 nm caused by

the disappearance of hydrogen peroxide1). The ε value at
240 nm for hydrogen peroxide was assumed to be
43.6 M–1 cm–1 14). The standard reaction mixture for the assay
contained 15 mM hydrogen peroxide, 3 μl of a catalase-containing solution and 50 mM phosphate buﬀer (pH 7.0), in a
total volume of 2 ml. The reaction was run for 1 min andthe initial linear rate was used to estimate the activity usinga V-550i spectrophotometer (JASCO International Co., Ltd.
Japan) equipped with a pen recorder. The amount of enzymethat decomposed 1 μmol of hydrogen peroxide per min was
defined as 1 U of activity. Protein concentration was determined using a protein assay kit purchased from Bio-Rad
laboratories with bovine serum albumin as a standard.
2.6. Sequence analysis of N-terminal amino acids
Protein SDS-PAGE was performed as described by
Laemmli19). Proteins were electroblotted from SDS-polyacrylamide gels onto a polyvinylidene difluoride (PVDF)
membrane (Clear Blot Membrane-P, ATTO Co., Ltd., Japan).
The digested band containing catalase was excised and used
for the amino acid sequence analysis. The N-terminal aminoacid sequence analysis was carried out by Toray ResearchCenter Inc., Japan. The N-terminal amino acid sequenceobtained was compared with those closely related bacteria
by homology search of the NCBI database.
2.7. Cloning of catalase gene
The molecular cloning of the catalase gene of strain 2-1
was performed by the PCR technique. The forward PCRprimer CatF: (5’-GAYATGAATAARAARCARGG-3’);(Y=C+T; R=A+G) was constructed on the basis of the
N-terminal amino acid sequence of the purified catalase derived from strain 2-1. The reverse primers, CatR1:( 5 ’ - R A A N A C N G G N GT RT T RT T N C C N A C - 3 ’ ) ;(N=A+T+G+C), CatR2: (5’-TGNGGCCANACYTTNGTNARRTCRAANGGTT-3’) were derived from the conserved region of the catalase of strain 2-1 and other catalases whose sequences were aligned using the tblastn
program of the DDBJ/GenBank/EMBL database. The total
chromosomal DNA was extracted from strain 2-1 using
ISOPLANT II kit (Nippon Gene Co., Ltd., Japan) and used
as a template. Touchdown PCR using Ex Taq polymerase(Takara Bio Co., Japan) was performed using an i-cycler(Bio-Rad Co., USA) with the following protocol: initialdenaturation at 94°C for 2 min, followed by 19 cycles ofdenaturation at 95°C for 20 s, annealing at 60°C for 20 s,
extension at 72°C for 1 min. The annealing temperature wasdecreased by 0.5°C at every subsequent set of cycles. Thiswas followed by 10 cycles of 20 s at 95°C, 20 s at 50°C and
40 s at 72°C, with a final extension of 4 min at 72°C. After
PCR, the total reaction mixture (20 μl) was separated byelectrophoresis in a 2.0% agarose gel and fragments of
0.4 kbp (CatF-CatR1) and 0.9 kbp (CatF-CatR2) were recovered and purified using the Geneclean III kit (Qbiogene,
Inc. USA).

Purification and cloning of catalase

37

Table 1. Purification of catalase from Rhizobium radiobacter strain 2-1.
Step

Volume (ml)Total Protein (mg)

Total Activity (U)Sp. activity(U/mg of protein)

Yield (%)

Purification (fold)

Crude Extract
Protein Denaturation
Ion Exchange
Gel Filtration

4.5
3.0
3.0
3.5

85.5
14.5
4.5
2.8

48163504128300
1376100
2140600

56331
284710305800764500

100
85
28
12

1
5
6
14

2.8. Determination of flanking nucleotide sequences ofcatalase using inverse PCR
Inverse PCR is a convenient and versatile method ofcloning unknown sequences upstream or downstream ofknown sequences without the constructing of genomic libraries11,26,29). The total chromosomal DNA of strain 2-1 was digested with Sal I and Pst I and self-ligated using T4 DNA
ligase (Takara bio Co.). Inverse PCR was carried out withthe forward primer invF (5’-GCCCACGGTCAGAGAGTTGCGGTCGCTTCA-3’) and the reverse primer invR1(5’-CGTGGCGAGCATCCGGCCTGGACGATGTCG-3’).
The PCR conditions were as follows: 94°C for 3 min, 98°C
for 10 s, 70°C for 5 min 30 s and 70°C for 10 min for 30 cycles.
The second inverse PCR was performed to obtain thedownstream side sequence of the same gene. The forward
primer (invF) and a newly constructed reverse primer invR2(5’-ACTGGTGCGCGAGGTGTTCAATGATGACGAG-3’)
were used to perform inverse PCR as described above exceptthat the number of cycles was 25.

Fig. 1. SDS-PAGE of purified catalase.
Lane 1, Molecular weight marker; lane 2, purified catalase.

2.9. DNA sequencing and data analysis
DNA sequencing was carried out using an ABI Prism 310
DNA sequencer (PE Biosystems, Foster City, USA). The
nucleotide and amino acid sequences were analyzed using
the GENETYX-MAC program ver. 8 (Software Development Co., Ltd., Tokyo, Japan), FASTA and BLAST on the
DDBJ/GenBank/EMBL nucleotide sequence databases.
Selected amino acid sequences were aligned using the Clustal
X program28).
3. Results

Fig. 2. Eﬀect of temperature on stability (A) and activity (B) of
catalase purified from strain 2-1.
For stability assay, the enzyme was incubated for 30 min at thegiven temperatures prior to the initiation of the reaction. Afterincubation, catalase activities were measured at 30°C, pH 7.
The activity is expressed as relative activity, taking the highest
activity observed as being 100%.

3.1. Catalase purification
A simple, three-step purification procedure consisting ofprotein denaturation, ion- exchange chromatography, and gel
permeation chromatography was developed to obtain purified
catalase from Rhizobium radiobacter strain 2-1. The resultsof each purification step are given in Table 1. The catalase waspurified 14-fold with a yield of 12% and a specific activity of
764,500 U/mg. SDS-PAGE analysis of the purified sample
confirmed to be a single band (Fig. 1). The molecular weightof the purified catalase was 56 kDa as determined by SDSPAGE. The molecular weight of the native catalase was also
estimated to be about 230 kDa by gel filtration using a
Superose 12HR10/30 column (data not shown). These observations indicate that the native catalase exists as a tetramer.

3.2. Optimal conditions for stability/activity of catalase
The catalase was stable within 30 min of incubation at
60°C, but was 40% inactivated at 65°C (Fig. 2A). The optimum temperature for the catalase was 30°C (Fig. 2B). The
pH for the optimum stability and activity of purified catalase
was also determined at 30°C. The catalase stability remained
almost the same at the pH range from 5 to 11 (Fig. 3A),
and the optimal pH range was also broad at 5 to 10 (Fig.
3B). The Km and Vmax of the purified catalase under optimum conditions were 60.06 mM and 10.17 mM/min, respectively.

HOSSAIN et al.

38

3.3. Cloning of catalase gene by PCR
The N-terminal amino acid sequence, TDMNKKQGGTGSTTGTGAPA, revealed from purified catalase ofstrain 2-1 was compared with those of the other catalase
genes by similarity search using the tblastn program on the
DDBJ/GenBank/EMBL database. It was revealed that the
N-terminal amino acid sequence showed 68% identity tothat of Nitrobacter hamburgensis strain X14 catalase.
Then, catalases showing high similarity to N. hamburgensis
X14 catalase were selected using the blastx program.
Among these, catalases from N. hamburgensis X14 andclosely related to R. radiobacter were selected and the aminoacid sequences were aligned. Consequently, two regions
where the sequences are highly conserved were identified(Table 2).
Touchdown PCR was performed using the forward primer
CatF and the reverse primers CatR1and CatR2, to amplify
the part of catalase gene from the strain 2-1 chromosomal
DNA. Consequently, PCR products of approximately 400 bp(CatF-CatR1) and 900 bp (CatF-CatR2) were obtained. The
nucleotide sequence of the CatF-CatR completely matchedwith the first half of the nucleotide sequence of CatF-CatR2. The size of this fragment roughly matched with thoseFig. 3. Eﬀect of pH on stability (A) and activity (B) of catalase
purified from strain 2-1.
The buffers (50 mM) used were sodium citrate (pH 4–6),
sodium phosphate (pH 6–7), Tris HCl (pH 8–9), GlycineNaOH (pH 9–10), Na2CO3-NaHCO3 (pH 10–11) and Na2PO4NaOH (pH 11–12). For stability assay, the enzyme wasincubated in each buﬀer at 30°C for 30 min. After incubation,
catalase activities were measured at 30°C, pH 7. For activity
assay, the enzyme activity was measured at 30°C in each of
the above buﬀers. The activity is expressed as relative activity,
taking the highest activity observed as being 100%of the corresponding regions of catalases used for the alignment.
To clone the entire length of the catalase gene, inverse
PCR was carried out on the basis of the partial nucleotide
sequence of the catalase mentioned above. An approximately 800 bp PCR product was amplified from the Sal I
digested and circulated chromosomal DNA of strain 2-1.
Sequence analysis suggested that the PCR product contained 80 bp of the upstream and 626 bp downstream regions of the putative catalase gene. The deduced N-terminal
amino acid sequence completely matched with that of thepurified catalase from strain 2-1. However, because the stopcodon was not found in the downstream side, inverse PCR
was performed again to determine the entire length of thecatalase gene. Using the reverse primer for inverse PCR(invR2) revealed an approximately 3.5 kbp fragment that
was amplified from the Pst I digested and circulated chromosomal DNA. Finally, a 351 bp downstream sequence including a putative stop codon was determined. The nucleotide
sequence of the cloned catalse gene, designated as katH,
have been submitted to GenBank and received the accession number AB370115.
3.4. Nucleotide sequence and deduced amino acid sequence
Figure 4 shows the complete nucleotide sequence of katHof strain 2-1. The resultant sequence contained one open
reading frame (ORF) consisting of 1,854 nucleotides, andthe N-terminal amino acid sequence (20 amino acids)
matched completely to that of the purifed catalase fromstrain 2-1. The stop codon TAA at the position 1555–1557
was followed by the stem-loop structure (5’-ACGCCGGCCCTCACAAGGGGCCGGCGT-3’), which is a putativetranscription termination point. The complete amino acidsequence of the catalase derived from strain 2-1 is also
shown in Fig. 4. This catalase (KatH) consists of 491 aminoacids with a molecular mass of 55059.39 Da. This result wasin good agreement with the molecular mass (56,000 Da) of
the purified catalase determined by SDS-PAGE (Fig. 1).
The deduced amino acid sequence was searched against the
GenBank database using the tblastx program3). It was foundthat R. radiobacter KatH showed more than 70% identity
to catalases from Sphingomonas wittichii RW1, Nitrobacter
hamburgensis, Thermobifida fusca and Micrococcus luteus(Table 3). In addition, the proximal active site was tenta-

Table 2. Alignment of amino acid sequences of catalases derived from strain 2-1 and closely related bacteria.
Species/Strain [Accession number]Conserved region

Strain 2-1
Nitrobacter hamburgensis X14[ABE61714]
Sinorhizobium meliloti [NP_384870](194) VGNNTPVF(131) VGNNTPVF(126) VGNNTPVF(343) NPFDLTKVWPH(280) NPFDLTKVWPH(275) NPFDLTKVWPH

Sinorhizobium meliloti [AAC44649](194) VGNNTPVF(343) NPFDLTKVWPH

Rhizobium sp. [AAA99823]
Streptomyces coelicolor A3(2)[CAC16511](123) VGNNTPVF(131) VGNNTPVF(272) NPFDLTKVWPH(280) NPFDLTKVWPH

Two highly conserved regions are shown. The number in the parentheses indicates the amino acids position.

Purification and cloning of catalase

39

Fig. 4. Complete nucleotide sequence of catalase gene from strain 2-1.
The double underline indicates the putative ribosome binding site located 7 bp upstream of the start codon. The stem loop (5’-ACGCCG
GCCCTCACAAGGGGCCGGCGT-3’) located downstream of the stop codon (TAA) is the putative transcription termination point. The
underlined parts indicate the inverse PCR primers. Bold letters indicate matching parts of the purified catalase amino acid sequence andthe N-terminal amino acid sequence.

Table 3. Amino acid homology analysis of catalase from strain 2-1.
Catalase gene

Homology

Sphingomonas wittichii RW1 [SwitDRAFT_2065]
Nitrobacter hamburgensis [Nham_0804]
Thermobifida fusca (strain YX) [Tfu_1649]
Micrococcus luteus (Micrococcus lysodeikticus) [katA]

83%
79%
74%
70%

HOSSAIN et al.

40

tively identified as residue 50–66 (apparent consensus
FNREKVPERQPHAKGSG) and the proximal heme ligand
was tentatively identified as residue 339–347 (apparent consensus RAFA YNDAQ).

pH range which makes this novel enzyme suitable for commercial exploitation and for removing hydrogen peroxide fromindustrial wastewater. Further studies are required to clarify therole of this catalase from the industrial perspective.

4. Discussion

References

The purification of catalase was performed using a highyielding catalase producing bacteria, Rhizobium radiobacterstrain 2-1. Although the amino acid sequence of KatH
showed more than 70% homology with the four putative
monofunctional heme catalases (Table 3), it showed no
more than 40% of the identities with other known R. radiobacter (Agrobacterium tumefaciens) strains (data notshown). Consequently, the present enzyme is a novel Rhizobium catalase, which can be classified as a monofunctionalheme catalase.
The molecular mass of a subunit of the catalase fromstrain 2-1 was estimated to be 56 kDa. The native molecularmass of the enzyme was estimated to be 230 kDa by gel
permeation chromatography, revealing that the catalaseconsists of four identical subunits. The subunit and native
molecular sizes for this enzyme are verily similar to thosereported for other tetrameric catalases (i.e., Bacillus sp.
with 70.5 and 282 kDa, Rhodobacter capsulatus with 59
and 236 kDa, T. brockianus with 42.5 kDa and 178 kDa,
and V. rumoiensis with 57.3 and 230 kDa, subunit andnative molecular sizes, respectively)31,37). The Km of thecatalase from strain 2-1 is 60.06 mM. This value is the sameas those of the reported thermostable catalases such as
from T. aurantiacus, T. brockianus, and B. subtilis (catalase-1 and catalase-2), whose Km values are 48, 35.5, 40.1
and 70 mM, respectively16,37). These observations strongly
suggest that this catalase is a typical catalase.
However, the specific activity of purified catalase fromstrain 2-1 was 143-fold higher than that of typical T. brockianus catalase, about 4-fold higher than that of M.luteus
catalase, and about 2-fold higher than that of V. rumoiensis catalase. These catalases are known for exhibiting highcatalase specific activity16,31,37).
In addition, the purified catalase from strain 2-1 showed
high temperature stability ranging from 4 to 60°C (Fig. 2A).
The activity also remained at about 40% at 65°C for
15 min, but the catalase was completely inactivated at 70°C.
Although the optimum reaction temperature of this catalase
was 30°C (Fig. 2B) and the activity decreased gradually athigher temperature, about 40% of the activity still remained
at 60°C. The purified catalase exhibited a broad pH stability
range (pH 5–11) and activity range (pH 5–10) with maximum activity at pH 6 (Fig. 3). There was no apparent substrate inhibition or inactivation of this purified catalase athydrogen peroxide concentrations up to 50 mM (data notshown). These results suggest that this catalase is suitablefor industrial use27).
From the commercial perspective, the purified catalase has
many beneficial properties such as high specific activity, simple
purification, high temperature stability and stability in a wide

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