Open access peer-reviewed chapter

Evolutionary Strategies of Highly Functional Catalases for Adaptation to High H2O2 Environments

Written By

Isao Yumoto, Yoshiko Hanaoka and Isao Hara

Submitted: October 23rd, 2020 Reviewed: December 14th, 2020 Published: January 11th, 2021

DOI: 10.5772/intechopen.95489

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Enzymatic evolutionary strategies for adaptation to a high H2O2 environment have been evaluated using catalases with high catalytic efficiency isolated from two H2O2-tolerant bacteria, Exiguobacterium oxidotolerans and Psychrobacter piscatori. The entrance size of the narrow main channel in catalase has been estimated by determining the formation rate of the intermediate state of peracetic acid (b), which is a larger substrate than H2O2 versus that of catalase activity with H2O2 (a) (calculated as b/a). The ratio of b/a in E. oxidotolerans catalase (EKTA) is much higher than that of P. piscatori catalase (PKTA). To elucidate the structural differences between the catalases, the amino acids present in the main channel have been compared between the two catalases and other catalases in the database. The combination of amino acid residues, which contribute high catalytic efficiency in the narrow main channel of EKTA were different from those in PKTA. In this review, we discuss strategic differences in the elimination of high concentration of H2O2 owing to differences in the phylogenetic positions of catalases. In addition, we describe the relationships between the environmental distributions of genera involved in H2O2-resistant bacteria and their catalase functions based on the main channel structure of catalase.


  • H2O2-tolerant bacteria
  • Exiguobacterium
  • Psychrobacter
  • Vibrio
  • catalase
  • narrow main channel
  • bottleneck size

1. Introduction

Oxygen is important for metabolism, acting as a terminal electron acceptor in aerobic bacteria, and these bacteria produce intracellular reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide (O2•−), and hydroxyl radical (OH) as by-products of oxygen metabolism [1, 2, 3, 4]. H2O2 is not a strongly harmful substance; however, the presence of H2O2 in bacterial cells may lead to the generation of harmful ROS, such as OH, via the Fenton reaction. Therefore, the presence of catalase is critical for the protection of cellular components, such as DNA, RNA, proteins, and lipids, from strongly harmful OH [5, 6, 7]. Moreover, the production of intracellular catalases is important for the metabolism of aerobic microorganisms to conduct their metabolisms.

Bacteria possess catalases for the elimination of toxic by-products of oxygen metabolism produced inside the cells and for preserving their niches by eliminating the H2O2 produced by host organisms [8, 9, 10]. This function is important, particularly for pathogenic and symbiotic microorganisms or microorganisms needing to maintain their niches in the host. In such cases, bacterial catalases may have evolved during interactions with the host (to degrade active oxygen species generated by the host for parasites elimination) or parasitic/symbiotic microorganisms (to eliminate active oxygen species generated by the parasites/symbionts). For example, Aliivibrio fischeri (formerly Vibrio fischeri) exhibits a symbiotic relationship with the host squid by colonising the light-emitting organs of the squid. The host squid possesses a protective mechanism associated with the production of H2O2 to prevent the colonization of unfavourable pathogenic bacteria. In contrast, A. fischeri produces highly efficient catalase in the periplasmic space to eliminate H2O2 produced by the host squid. Thus, production of catalase in the vicinity of the cell surface is important for helping microorganisms to establish their niche.

The oral biofilm community consists of various microorganisms, including foe and companion bacteria and functions to maintain the ecological balance among constituents [11]. Among these community members, Streptococcus gordonii is known to produce H2O2 to expel its competitors. Additionally, Veillonella atypica is able to support the growth of the obligate anaerobe, Fusobacterium nucleatum under microaerophilic conditions and can also protect the microorganism from S. gordonii via production of catalase. Thus, extracellular catalase production is important for protection not only of the niche of the producer but also of other companion microorganisms to facilitate the formation of microbial communities within biofilm.

Catalase is commonly observed in various aerobic bacteria. Bacteria that do not possess catalase cannot grow on the agar plates owing to the presence of H2O2 on agar plates [12]. However, many bacterial strains have been isolated from agar plates, suggesting that these bacteria likely express catalase and these bacteria are likely to encounter H2O2. Moreover, these data suggest that H2O2 may be ubiquitously present in various environments in which many microorganisms live. Accordingly, investigation of the molecular strategies through which catalase eliminates H2O2 in various physiological, ecological, and taxonomic background is essential.

In this review, we evaluate the relationships between catalase evolution and structural changes in the main channel structure of catalases, based on various catalases including those isolated from H2O2- tolerant bacteria. In addition, considering the taxonomic backgrounds of H2O2-tolerant bacteria, we compared the main channel structures of catalases derived from the same genera of H2O2-tolerant bacteria and discussed the reasons for the distribution of these H2O2-tolerant bacteria. This systematic approach will bring deeper understanding in strategic evolutionary changes in bacterial catalases and strategic bacterial distributions in the environment.


2. Phylogeny of catalases

The dismutation of H2O2 in microorganisms occurs mainly via three phylogenetically unrelated catalases: monofunctional catalase, catalase-peroxidase, and Mn-catalase [2, 13]. Here, we focus on monofunctional catalases.

Bacterial monofunctional catalases are classified into clades 1–3 according to phylogenetic analysis based on their amino acid sequences [14, 15]. Clade 1 catalases contain approximately 500 amino acid residues per subunit and are mainly of plant origin, except a subgroup that is of bacterial origin, including Firmicutes group A and Proteobacterial minor group (Sinorhizobium clade). Clade 2 catalases, which exhibit larger molecular masses than catalases from other clades, consist of approximately 750 amino acid residues. The catalases in this clade originated from fungi, bacteria including Actionbacteria, Bacteroides, and Proteobacteria (Polaromonas, Burkholderia and Akkermansia) and archaea. Clade 3 catalases, with nearly 500 residues per subunit, occur in fungi, bacteria including Chloroflexi, Firmicutes group B and Proteobacteria, fungi, and some eukaryotes. Reports have shown that pathogenic or symbiotic bacteria possess only one clade 3 catalase (e.g., Haemophilus influenzae, Neiseria gonorrhoeae, and A. fischeri [described above]). These catalases evolved through interactions between the host and parasite. Moreover, many prokaryotic clade 3 catalases exhibit distinct NADP(H) binding compared with clade 1 catalases, discrimination of catalases between the two clades based on apparent molecular features and enzymatic characteristics is difficult.


3. Reaction mechanisms of catalases

Catalase consists of four identical subunits and each subunit, each of which possesses heme b or d at the reaction centre. The catalytic reaction cycle consists of the following two steps. The first step involves the formation of compound I, which is produced by oxidation of Fe3+ (Fe3+ Pro) in the heme moiety to an oxoiron (IV) porphyrin π-cation radical species, Fe4+ = O Pro+•, by the first reacted H2O2 molecule [16]. During this reaction, the oxygen–oxygen bond in the peroxide (R–O–O–H) bound to the heme, that is the first H2O2 molecule, is cleaved heterolytically. As a result, one oxygen binds to the ion with the by-product of a water molecule. This reaction intermediate, compound I, is subsequently reduced by second reaction of H2O2 to the resting state (Fe3+ Pro). This reaction leads to the production of molecular oxygen (O2) and water molecules (H2O) [17, 18]. Compound I can also be observed if organic peroxides are used as substrates instead of H2O2. The compound I formation rate decreases as the molecular size of the substrates increases (i.e., H2O2 ˃ CH3COO2H). Therefore, estimation of the compound I formation rate may be an indicator of the size of the bottleneck structure of the narrow main channel, which is directly accessible to the reaction centre, heme.


4. Characteristics of H2O2-resistant bacteria

Catalase is important for cellular protection intra- and extracellular elimination of H2O2. Because H2O2-tolerant microorganisms may evolve in artificially high H2O2 environments, we have studied H2O2-tolerant microorganisms and their catalases. First, strain S-1T can survive downstream of drain pools (sedimentation tank [8°C, 1.5–6 mM]) from herring egg processing factory, which uses H2O2 as a breaching agent [19, 20]. This strain was identified as a new species, Vibrio rumoiensis S-1T. The growth temperature range of strain S-1T is 2–34°C. The catalase activity of cell extracts of strain S-1T was found to be 4000–8000 U/mg protein, which was one or two orders of magnitude higher than those of Alcaligenes faecalis, Corynebacterium glutamicum, and Pseudomonas fluorescens. Strain S-1T possesses only one type of clade 3 catalase, which accounts for 1.8% of the protein in cell extracts. The isolate produces catalase not only inside the cell but also in the periplasmic space and on the cell surface [21, 22, 23, 24]. Therefore, V. rumoiensis S-1T cells exhibit catalase activity, and expression of catalase on the surface of V. rumoiensis cells may help to protect the cell in high H2O2 environments. According to several reports on symbiotic or pathogenic strains involving the genus Vibrio and its related genus Aliivibrio, strain S-1T was predicted to be derived from marine environments or organisms.

Strain T-2-2T, an H2O2-tolerant microorganism, was isolated from the upstream region of a water treatment system (pretreatment tank to decrease H2O2 concentration [8°C, 6–38 mM]) of a herring egg processing factory [25]. The isolate was identified as a new species, E. oxidotolerans T-2-2T. The growth temperature range of this strain was 4–40°C (optimum 34°C). The cell extract of strain T-2-2T exhibited catalase activity of 28,000 U/mg protein and catalase accounted for 6.5% of protein in the cell extract. The bacterium produced catalase (E. oxidotolerans [EKTA]) both intercellularly and extracellularly [26, 27, 28, 29]. The immunolocalization of catalase suggests that the enzyme is present on the inner surface of the cells [28]. Catalase that bind to the cell surface and localise to the inner surface are also important for defence against extracellular H2O2 in E. oxidotolerans T-2-2T. The localisation of catalase changes from inside of the cells to the cell surface as the culture period is extended. The catalase is induced by H2O2 stimulation prior to initiation of growth and low aeration growth condition [27, 29]. Thus, catalase activity is required inside the cells and is essential for extracellular defence as the cell age increases. Exiguobacterium spp. are distributed in various environments, including marine environments [30, 31]. Therefore, strain T-2-2T may have originated from marine environments or organisms. Additionally, although strain T-2-2T possesses a catalase gene sequence belonging to clade 2, only clade 1 catalase can be purified [32].

Strain T-3-2T, an H2O2-tolerant microorganism, was isolated from the upstream of the water treatment system (pretreatment tank to decrease H2O2 concentration [8°C, 6–38 mM]) of a herring egg processing factory [33]. The growth temperature range of strain T-3-2T is 0–30°C, and the localisation of catalase has not yet been clarified. However, strain T-3-2T exhibits high resistance against H2O2. The isolate was identified as a new species, P. piscatorii T-3-2T and cell extracts of strain T-3-2T exhibit much higher catalase activity (12,000 U/mg protein) than those of other stains belonging to the same genus, including Psychrobacter nivimaris (15 U/ mg protein), Psychrobacter proteolyticus (29 U/mg protein) and Psychrobacter aquamaris (1800 U/mg protein). Strain T-3 belongs to P. piscatorii as well [34, 35] and exhibits higher catalase activity (19,700 U/mg), with catalase accounting for 10% of all proteins in the cell extract. Several reports have described Psychrobacter spp. were isolated from marine origins [36]; therefore, it is possible that strains T-3-2T and T-3 originated from marine environments or organisms. Although the strain T-3 possesses catalase gene sequences belonging to clade 2, only clade 3 catalase can be purified [32].


5. Characteristics of catalases from H2O2-resistant bacteria

Catalases derived from H2O2-tolerant microorganisms in clade 3 and clade 1 have been purified from V. rumoiensis S-1T, P. piscatorii T-3, and from E. oxidotolerans T-2-2T. The kinetic parameters (kcat/Km) of these catalases were higher or equivalent to the highest values comparing with those of catalases reported by Switala and Loewen (2002) [37]. In addition, these catalase activities exhibited distinctive temperature dependencies comparing with ordinary catalase such as Micrococcus luteus catalase (MLC) and bovine liver catalase (BLC) [32]. These characteristics reflect the environmental conditions in which these bacteria were isolated (8°C, 1.5–38 mM H2O2). Thus, multiple environmental factors (including low temperature and high H2O2) have affected the characteristics of enzymes via evolutionary and/or environmental selection processes.

The catalase from V. rumoiensis S-1T (VKTA) can be purified by two steps of anion-chromatography and one step of gel filtration chromatography [25]. The purified VKTA exhibits 395,000 U/mg protein under standard reaction conditions (30 mM H2O2, pH 7), with a Vmax and Km of 8.0 × 105 μmol H2O2/μmol heme/s and 35 mM for H2O2, respectively, as determined spectrophotometrically. The catalytic efficiency kcat/Km of VKTA is 2.3 × 107/s/M, which is the highest among reported clade 3 catalases owing to the low Km value [31]. Additionally, because of the fragility of V. rumoiensis S-1T cells, high affinity to H2O2 and high catalytic efficiency are required for protection of the cells. It is known that catalase activity is not as dependent on temperature as the activity of ordinary enzymes. Moreover, VKTA exhibits an obvious temperature dependence between 10°C and 70°C with an optimum temperature at 40°C. The amino acid sequence of VKTA contains active sites (H61, T100 and N134), proximal sites of heme (Y344 and R351), and binding sites for the distal region of heme (V102, T124 and F139). VKTA possesses NADPH- binding sites (H180, R189, V288 and K291). The active site containing “T100” is unique compared with that of the other catalases listed in Figure 1. Indeed, other catalases contain an “S residue at this position”, making the site less hydrophobic. However, the effect of this amino acid substitution on the function is unknown.

Figure 1.

Amino acid sequence alignment of EKTA, Exiguobacterium enclense catalase, Exiguobacterium aurantiacum catalase, Listeria innocua catalase, Deinococcus radiodurans KatA, PSCF, MLC, Proteus mirabilis catalase, Aliivibrio salmonicida (VSC) catalase, Vibrio halioticoli catalase, VKTA, Psychrobacter phenylpyruvicus catalase and Psychrobacter cryohalolentis catalase. The amino acid residues involved in the narrow main channel are highlighted by green or yellow (bottleneck residues). The active sites are indicated in bold font, the proximal sites of heme are marked in blue and the binding sites of distal region of heme are marked by underlined text.

EKTA can be purified by two steps of anion-chromatography and one step of gel filtration chromatography. The purified EKTA exhibits an activity of 430,000 U/mg protein under standard reaction condition [26] with a Vmax and Km of 1.5 × 106 μmol H2O2/μmol heme/s and 40 mM for H2O2, respectively, as determined by spectrophotometry [28]. The catalytic efficiency kcat/Km of EKTA is 3.8 × 107/s/M, which is the highest among reported clade 1 catalases owing to the high kcat and low Km values. EKTA exhibits a temperature dependency between 10°C and 70°C with an optimum temperature of 45°C. Catalase activity decreases from 100–60% as the temperature increases from 45–50°C and then is further decreased to approximately 10% at 70°C. Moreover, this catalase exhibits the highest temperature sensitivity among the three catalases purified from the three H2O2- tolerant bacteria. The amino acid sequence of EKTA contains active sites (H56, S104 and N138), proximal sites of heme (Y339 and R346) and binding sites for the distal region of heme (V97, T119 and F142), as shown in Figure 1. There is no NADPH-binding site in the amino acid sequence of this catalase. These important residues for catalase activity are well conserved in EKTA.

The catalase from P. piscatorii T-3 (PKTA) can be purified by one step of anion-chromatography and one step of hydrophobic chromatography [35]. The purified PKTA exhibits an activity of 222,000 U/mg protein under standard reaction conditions, with Vmax and Km of 2.4 × 105 μmol H2O2/μmol heme/s and 75 mM for H2O2, respectively. The catalytic efficiency kcat/Km of PKTA is 3.2 × 106/s/M as determined with O2 electrode [34]. PKTA exhibits a temperature dependency between 10°C and 80°C with an optimum temperature of 45°C. The activity decreases at temperature over 50°C, showing approximately 10% at 70°C and complete deactivation at 85°C. The amino acid sequence of PKTA contains active sites (H65, S104 and N138), proximal sites of heme (Y348 and R355), and binding sites for the distal region of heme (V106, T128 and F143). This PKTA also contains NADPH-binding sites (H184, R193, V292 and K295), as shown in Figure 1. These important residues for catalase activity are well conserved in PKTA.


6. Relationship between the compound formation rate with peracetic acid and the bottle neck amino acid residue in the narrow main channel

Catalase is known to have high activity owing to its superior substrate selectivity for H2O2. The interactions of substrate molecules larger than H2O2 are strongly inhibited due to selection of the substrate by the narrow main channel, which reaches the active site. The formation rate of the reactive intermediate (compound I) in the reaction of EKTA with peracetic acid is 77 times higher than that of BLC and 1200 times higher than that of MLC [26]. A comparison of the structural and functional data on EKTA (a clade 1 catalase) with the data for two clade 3 catalases (BLC and MLC) revealed that the size of the bottleneck defines the compound I formation rate, which corresponds to the size of the substrate molecule. The atom-to-atom distance for combinations of amino acid residues showed that, the L149 (BN [bottleneck] 2) to I180 (BN4) and D109 (BN1) to M167 (BN3) combinations at the bottleneck of EKTA resulted in larger bottleneck sizes than the combinations in BLC and MLC [26]. The sizes of the amino acids and the probability of occurrence of the corresponding amino acids (based on a comparison of catalase sequences in the database) indicated that M167 may play a key role in determining the size of the bottleneck of EKTA. Clade 3 catalases, i.e., BLC and MLC contain W (Phe) in the corresponding position of M167 in EKTA. The volume of W (Phe) is 231.7 Å3, whereas that of M (Met) is 167.7 Å3 [38, 39]. Therefore, the size of the key residue M167 in EKTA is the major reason for the high the compound I formation rate with peracetic acid.


7. Comparison of amino acid residues in the narrow main channel of catalase

The main channel of catalase consists upper and lower narrow parts. The narrow part, which is nearer to the reaction centre, heme consists of 14 amino acid residues [26] (Figures 1 and 2). The seven residues forming the channel (H56, V97, D109, N129, F134, F135 and F142 in EKTA) are well conserved (≥95% homology). V55 is relatively highly conserved (≥ 80%) followed by P110 (54%). The other amino acid residues, including M145 (approximately 20%), V146 (approximately 30%) and L149 (approximately 20%) are relatively rarely conserved. Both M167 and I180 are very rarely conserved (≤ 3%) among catalases. Among the 14 amino acid residues described above, D109 (BN1), L149 (BN2), M167 (BN3) and I180 (BN4) are located in the bottleneck structure in between the upper and lower parts of the main channel of catalase. Among these four amino acid residues only D109 is well conserved. Therefore, variations in amino acid residues, except D109, define the size of the bottleneck structure and the reaction rate with substrates larger than H2O2. Based on the alignment of multiple catalases including other catalases derived from other species belonging to the genus Exiguobacterium, there are several common amino acid residues between EKTA and Exiguobacterium enclense catalase (M145, V146, L149 and M167). Owing to the lower volumes of these residues compared with the corresponding residues in other catalases, these residues are thought to be related to the genus-specific efficiency catalytic reactions in the presence of high concentrations of H2O2.

Figure 2.

Structural model of narrow main channels of catalases of VSC [A] and EKTA [B]. Each characteristic amino acid residues are indicated by yellow marker. Number for amino acid residues was accordance with Figure 1. The amino acid residues of narrow main channels in VSC are the same as VKTA. aThis amino acid residue is substituted to “T” in PKTA. bThis amino acid residue is modified as S-dioxymethionine.

In contrast, M 64, I119, L154, N155 and T158 are specific amino acid residues in the narrow main channel of PKTA. These amino acid residues are corresponding to M60, I115, L150, N151 and V154 in VKTA and catalases from Proteus mirabilis, Aliivibrio salmonicida, Psychrobacter phenylpyruvicus and Psychrobacter cryohalolentis catalases. Although the activities of the latter two catalases are not known, the other three catalases exhibit high catalytic efficiency for H2O2 [40]. Therefore, these residues are specific to the catalase of Proteobacteria and affect the efficiency of these catalases.


8. Relationship between catalase phylogeny and the main channel structure of catalases

The clade 1 catalase EKTA exhibits a higher ratio (b/a = 1.4) of the compound I formation rate using peracetic acid (a) to catalase activity using H2O2 (b) than the clade 3 catalase PKTA (b/a = 0.0056) [29]. Although the size of the bottleneck of PKTA is unknown, the difference in the catalytic characteristics can be attributed to the seize of the bottleneck, which this can be ascertained from the amino acid residues in the bottleneck. In addition to EKTA and PKTA, the b/a ratio was estimated using the clade I catalases, Pseudomonas syringae catalase (PSCF) and Deinococcus radiodurans catalase and the clade 3 catalases BLC and MLC. Differences in the b/a ratio are related to the intensity of the degree of the extended branch in the phylogenetic tree of catalase (Table 1 and Figure 3). This indicates that catalases from H2O2-tolerant bacteria evolved in different directions depending on the bacterial taxonomic phylogenetic position. Thus, the phylogenetic position can be ascertained based on the amino acid sequences of catalase from Exiguobacterium spp. and Psychrobacter spp. However, it has been difficult to discriminate clade 1 and clade 3 catalase except phylogenetic position based on amino acid sequences. Indeed, these catalases can be discriminated based on differences in the catalytic efficiency for H2O2 according to the structure of the narrow main channel.

Bottle neck structure
BN2–BN4L149, M167, I180V158, W176, T189V154, W172, V185
The size of BN2–BN4164.6, 167.7, 164.9150.6, 231.7, 120.0150.6, 231.7, 139.1
Enzymatic feature
b/a ratioa1.40.0056ND
Kinetic parameters for H2O2
Vmax (/s)1.5 × 106b2.4 × 105c8.0 × 105b
Km (mM)40b75c35b
kcat/Km (/M/s)3.8 × 107b3.2 × 106c2.3 × 107b
Cellular features
Percentage of catalase in cell extract6.5%10%1.8%
Location of isolationUpstream of the drain (6–38 mM H2O2)Upstream of the drain (6–38 mM H2O2)Downstream of the drain (1.5–6 mM H2O2)
Involved bacteriaGram positiveGram negativeGram negative
Extended of phylogenetic positionYesYesYes
Purified catalase activity (U/mg)c
Catalase activity of cell extract (U/mg)d28,00020,0007,300

Table 1.

Summary of the characteristics of catalases from H2O2-tolerant bacteria.

The ratio of compound I formation rate using peracetic acid (a) to catalase activity using H2O2 (b).

Determined by spectrophotometry.

Determined by oxygen electrode analysis.

Standard reaction conditions of 30 mM H2O2 at pH 7.

Figure 3.

Phylogenetic position of catalases clades 1–3. The phylogenetic tree was constructed using the Maximum Likelihood method and JTT matrix-based model [41]. Multiple alignments of the sequences were performed using the MUSCLE program [42]. The numbers in the branches indicate bootstrap percentages based on 500 replicates. Bar, 0.20 changes per amino acid position. Evolutionary analyses were conducted in MEGA X [13].


9. Environmental distribution and catalase function of H2O2-resistant bacteria

Results of a screening of bacterial strains adapted to high H2O2 environments (8°C, 6–38 mM H2O2), E. oxidotolerans T-2-2T and P. piscatorii T-3T and T-3-2 were isolated. Some microorganisms have been shown to thrive under extreme environments such as high and low temperatures and high and low pH. However, Exiguobacterium spp. and Psychrobacter spp. are known widely distributed in polar regions, permafrost, deep sea regions, temperate and tropical soils, and ordinary marine environments [43, 44]. Therefore, several strains belonging to the genera Exiguobacterium and Psychrobacter have been identified as psychrophilic or psychrotolerant bacteria. In addition to the cold-adapted variations of these genera, our studies revealed that there were variations in the H2O2 tolerance of these genera.

Although these genera exhibit common physiological characteristics and environmental distributions, phylogenetic positions are completely different from a taxonomical point of view [43]. Gram-positive Exiguobacterium belongs to the phylum Firmicutes, class Bacilli, and order Bacillales, whereas Psychrobacter belongs to the phylum Proteobacteria, class Gammaproteobacteria, order Pseudomonadales, family Moraxellaceae. Dias et al. analysed and compared four genomes of Exiguobacterium and Psychrobacter [44] and showed that Psychrobacter exhibited higher genomic plasticity, whereas E. antarcticum exhibited a large decrease in genomic content without changing its adaptability to cold environments. These results suggest that the H2O2 tolerance and molecular features of catalases and their productivities in H2O2-tolerant bacteria belonging to Exiguobacterium and Psychrobacter were related to the intrinsic genomic architectural dynamics of these taxa.

V. rumoiensis was isolated from an environment containing lower H2O2 concentration (1.5–6 mM) than the other two strains. The genus Vibrio and the closely related genus Aliivibrio are known for involving species of their pathogenicity and symbiosis with marine organisms. Thus, these organisms may have high capacity for adaptability to high H2O2 environment. Moreover, bacterial genome analysis of six bacterial species belonging to the rumoiensis clade revealed that there are ecogenomic signatures inferring the ongoing habit expansion in two strains (V. rumoiensis included) [45]. Thus, this microorganism may have adapted to environments containing high H2O2 by genomic altering specific characteristics.


10. Conclusion and future studies

It has been shown that completely different taxa of bacteria evolve catalases in different directions improving productivity of catalases in the same or similar environment (i.e., low temperature and high H2O2 concentration). Adaptations to environments with high concentration of H2O2 has been achieved by certain groups of bacteria, including psychrotolerant bacteria originating from marine environments, which are widely distributed and can survive under various environmental conditions (e.g., low temperature and high H2O2 concentration). This adaptability is observed in terms of enzymatic features, productivity and localisation of catalase. Future studies are necessary to analyze the evolutionary process in more detail and determine the relationship of this evolutionary process with the functions of specific enzymes. Furthermore, detailed studies of the microbiota present in environments containing high H2O2 concentrations may provide insight into the mechanisms through which bacteria adapt to artificial extreme environments.


We greatly thank Professor Hidetoshi Matsuyama for his continues support in our study. We wish to thank Mr. Daisen Ichihashi, Mr. Hideaki Iwata, Mr. Fumihiko Takebe, and Mr. Hideyuki Kimoto for providing technical assistance.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Appendices and nomenclature

Website resources:

Catalase website (

EMBEL-EBI Catalase


Catalase (enzyme nomenclature designation [EC]


  1. 1. Bolton JL, Trush MA, Penning TM, Dryhurt G, Monks TJ: Role of quinones in toxicology. Chem Res Toxicol. 2000;13:135-160.
  2. 2. Lin P-C, Türk K, Häse CC, Fritz G, Steuber J: Quinone reduction by the Na+−translocating NADH dehydrogenase promotes extracellular superoxide production in Vibrio cholerae. J Bacteriol. 2007; 189:3902-3908. doi: 10.1128/JB.01651-06
  3. 3. Treberg JR, Quinlan CL, Brand MD: Evidence for two sites of superoxide production by mitochondrial NADH-ubiquinone oxidoreductase (Complex I). J Biol Chem. 2011;286:27103-27110.
  4. 4. Yin Y, Yang S, Yu L, Yu CA: Reaction mechanism of superoxide generation during ubiquinol oxidation by the bc1 complex. J Biol Chem. 2011;285:17038-17045.
  5. 5. Halliwell B, Gutteridge JMC Free Radical in Biology and Medicine, 3rd ed. Clarendon Press, 1999.
  6. 6. Imlay JA, Linn S: DNA damage and oxygen radical toxicity. Science. 1988;240:1302-1309.
  7. 7. Rowe LA, Degtyareva N, Doetsch PW : DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae. Free Radic Biol Med. 2008;45:1167-1177.
  8. 8. Katsuwon J, Anderson AJ: Characterization of catalase activities in root colonizing isolates of Pseudomonas putida. Can J Microbiol. 1992;38:1026-1032.
  9. 9. Rocha ER, Selby T, Coleman JP, Smith CJ: Oxidative stress response in an anaerobe, Bacteroides fragilis: A role for catalase in protection against hydrogen peroxide. J Bacteriol. 1996;178:6895-6903.
  10. 10. Visick KL, Ruby EG: The periplasmic, group III catalase Vibrio fischeri is required for normal symbiotic competence and is induced both by oxidative stress and by approach to stationary phase. J Bacteriol. 1998;180:2087-2092.
  11. 11. Zhou P, Li X, Huang I-H, Qi F: Veillonella catalase protects the growth of Fusibacterium nucleatum in microaerophilic and Streptococcus gordonii-resistant environments. Appl Environ Microbiol. 2017;83:e01079–e01017.
  12. 12. Tanaka T, Kawasaki K, Daimon S, Kitagawa W, Yamamoto K, Tamaki H, Tanaka M, Nakatsu CH, Kamagata Y: A hidden pitfall in preparation of agar media undermines microorganism cultivability. Appl Environ Micobiol. 2014;80:7659-7666.
  13. 13. Kumar S, Stecher G, Li M, Knyaz C, Tamura K; MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2008; 35:1547-1549.
  14. 14. Klotz MG, Klassen GR, Loewen PC: Phylogenetic relationships among prokaryotic and eukaryotic catalases. Mol Biol Evol. 1997;14:951-958.
  15. 15. Zámocký M, Gasselhuber B, Furtmuller PG, Obinger C: Molecular evolution of hydrogen peroxide degrading enzymes. Arch Biochem Biophys. 2012;525:131-144.
  16. 16. Jones P, Middlemiss DN: Formation of compound I by the reaction of catalase with peroxoacetic acid. Biochem J. 1972;130:411-415.
  17. 17. Deisseroth A and Dounce AL: Catalase: Physical and chemical properties, mechanism of catalysis, and physiological role. Physiol Rev.1970;50:319-375.
  18. 18. Schonbaum GR, Chance B Catalase, in The enzymes (Boyer, P. D., Ed.) Academic Press; 1976. p 363-408.
  19. 19. Yumoto I, Yamazaki K, Kawasaki K, Ichise N, Morita N, Hoshino T, Okuyama H: Isolation of Vibrio sp. S-1 exhibiting extraordinarily high catalase activity. J Ferment Bioeng. 1998;85:113-116.
  20. 20. Yumoto I, Iwata H, Sawabe T, Ueno K, Ichise N, Matsuyama H, Okuyama H, Kawasaki K: Characterization of a facultatively psychrophilic bacterium, Vibrio rumoiensis sp. nov., that exhibits high catalase activity. Appl Environ Microbiol. 1999;65:67-72.
  21. 21. Ichise N, Morita N, Hoshino T, Kawasaki K, Yumoto I, Okuyama H: A mechanism of resistance to hydrogen peroxide in Vibrio rumoiensis S-1. Appl. Environ. Microbiol. 1999;65:73-79.
  22. 22. Ichise N, Morita N, Kawasaki K, Yumoto I, Okuyama H; Gene cloning and expression of the catalase from the hydrogen peroxide-resistant bacterium Vibrio rumoiensis S-1 and its subcellular localization. J Biosci Bioeng. 2000;90:530-534.
  23. 23. Ichise N, Hirota K, Ichihashi D, Nodasaka Y, Morita N, Okuyama H, Yumoto I; H2O2 tolerance of Vibrio rumoiensis S-1T is attributable to the cellular catalase activity. J Biosci Bioeng. 2008;106:39-45.
  24. 24. Yumoto I, Ichihashi D, Iwata H, Istokovics A, Ichise N, Matsuyama H, Okuyama H, Kawasaki K; Purification and characterization of a catalase from the facultative psychrophilic bacterium Vibrio rumoiensis S-1T exhibiting high catalase activity. J Bacteriol. 2000;182:1903-1909.
  25. 25. Yumoto I, Hishinuma-Narisawa M, Hirota K, Shingyo T, Takebe F, Nodasaka Y, Matsuyama H, Hara I; Exiguobacterium oxidotolerans sp. nov., a novel alkaliphile exhibiting high catalase activity. Int J Syst Evol Microbiol. 2004;54:2013-2017.
  26. 26. Hara I, Ichise N, Kojima K, Kondo H, Ohgiya S, Matsuyama H, Yumoto I; Relationship between the size of the bottleneck 15 Å away from iron in the main channel and reactivity of catalase corresponding to the molecular size of substrates. Biochemistry. 2007;46:11-22.
  27. 27. Takebe F, Hara I, Matsuyama H, Yumoto I; Effect of H2O2 under low- and high-aeration-level conditions on growth and catalase activity in Exiguobacterium oxidotolerans T-2-2T. J Biosci Bioeng. 2007;104:464-469.
  28. 28. Hanaoka Y, Takebe F, Nodasaka Y, Hara I, Matsuyama H, Yumoto I; Growth-dependent catalase localization in Exiguobacterium oxidotolerans T-2-2T reflected by catalase activity of cells. PLoS ONE. 2013;8:e76862.
  29. 29. Hanaoka Y, Yumoto I; Manipulation of culture conditions for extensive extracellular catalase production by Exiguobacterium oxidotolerans T-2-2T Ann Microbiol. 2015;65:1183-1187.
  30. 30. Dastager SG, Mawlankar R, Sonalkar VV, Thorat MN, Mual P, Verma A, Krishnamurthi S, Tang SK, Li WJ; Exiguobacterium enclense sp. nov., isolated from sediment. Int J Syst Evol Microbiol. 2015; 65:1611-1616. doi: 10.1099/ijs.0.000149.
  31. 31. Lei F, Cui C, Zhao H, Tang X, Zhao M; Phylogenetic diversity and biotechnological potentials of marine bacteria from continental slope of eastern Arabian Sea. J Genet Eng Biotechnol. 2018; 16:253-258. doi: 10.1016/j.jgeb.2018.06.002.
  32. 32. Yumoto I, Hara I Psychrotolerant bacteria and environmental H2O2-resistant adaptation of their catalases. In: Yumoto I (ed) Cold-adapted microorganisms. Caister Academic Press; 2013. p 137-158.
  33. 33. Yumoto I, Hirota K, Kimoto H, Nodasaka Y, Matsuyama H, Yoshimune K; Psychrobacter piscatorii sp. nov., a psychrotolerant bacterium exhibiting high catalase activity isolated from an oxidative environment. Int J Syst Evol Microbiol. 2010;60:205-208.
  34. 34. Kimoto H, Matsuyama H, Yumoto I, Yoshimune K; Heme content of recombinant catalase from Psychrobacter sp. T-3 altered by host Escherichia coli growth condition. Protein Express Purif. 2008;59: 357-359.
  35. 35. Kimoto H, Yoshimune K, Matsuyama H, Yumoto I; Characterization of catalase from psychrotolerant Psychrobacter piscatorii T-3 exhibiting high catalase activity. Int J Mol Sci. 2012;13:1733-1746.
  36. 36. Romanenko LA, Schumann P, Rohde M, Lysenko AM, Mikhailov VV, Stackebrandt E; Psychrobacter submarinus sp. nov. and Psychrobacter marincola sp. nov., psychrophilic halophiles from marine environments. Int J Syst Evol Microbiol. 2002;52:1291-1297. doi: 10.1099/00207713-52-4-1291.
  37. 37. Switala J, Loewen PC; Diversity of properties among catalases. Arch Biochem Biophys. 2002; 401:145-154.
  38. 38. Harpaz Y, Gerstein M, Chothia C; Volume changes on protein folding. Structure. 1994;15:641-649.
  39. 39. Hanaoka Y, Kimoto H, Yoshimune K, Hara I, Matsuyama H, Yumoto I; Relationship between main channel structure of catalases and the evolutional direction in cold adapted hydrogen peroxide-tolerant Exiguobacterium and Psychrobacter. Indian J Microbiol. 2020;60:353-362.
  40. 40. Lorentzen MS, Moe E, Jouve HM, Willassen NP; Cold adapted feature of Vibrio salmonicida catalase: characterization and comparison to the mesophilic counterpart from Proteus mirabilis. Extremophiles. 2006;10:427-440.
  41. 41. Jones D.T., Taylor W.R., and Thornton J.M.; The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8: 275-282.
  42. 42. Edgar RC; A multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5:113.
  43. 43. Rodrigues DF, da C Jesus E, Ayala-Del-Río HL, Pellizari VH, Gilichinsky D, Sepulveda-Torres L, Tiedje JM; Biogeography of two cold-adapted genera: Psychrobacter and Exiguobacterium. ISME J. 2009; 3:658-665.
  44. 44. Dias LM, Folador ARC, Oliveira AM, Ramos RTJ, Silva A, Baraúna RA; Genomic architecture of the two cold-adapted genera Exguibacterium and Psychrobacter: evidence of functional reduction in the Exgiguobacterium antarcticum B7 genome. Genome Biol Evol. 2018;10:731-741.
  45. 45. Tanaka M, Kumakura D, Mino S, Doi H, Ogura Y, Hayashi T, Yumoto I, Cai M, Zhou Y.-G, Gomez-Gil B, Araki T, Sawabe T; Genomic characterization of closely related species in the Rumoiensis clade infers ecogenomic signatures to non-marine environments. Environ Microbiol. 2020; 22: 3205-3217.

Written By

Isao Yumoto, Yoshiko Hanaoka and Isao Hara

Submitted: October 23rd, 2020 Reviewed: December 14th, 2020 Published: January 11th, 2021