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

*Isao Yumoto, Yoshiko Hanaoka and Isao Hara*

## **Abstract**

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.

**Keywords:** 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 [1–4]. 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• [5–7]. 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 [8–10]. 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

**505**

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

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

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,

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

. The growth temperature range of strain S-1T

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

tion 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,

can survive downstream of drain pools (sedimenta-

was found to be 4000–8000 U/mg

is 2–34°C. The

*DOI: http://dx.doi.org/10.5772/intechopen.95489*

characteristics is difficult.

**3. Reaction mechanisms of catalases**

which is directly accessible to the reaction centre, heme.

**4. Characteristics of H2O2-resistant bacteria**

catalase activity of cell extracts of strain S-1T

catalases. First, strain S-1T

*Vibrio rumoiensis* S-1T

*Evolutionary Strategies of Highly Functional Catalases for Adaptation to High H2O2 Environments DOI: http://dx.doi.org/10.5772/intechopen.95489*

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**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

important for helping microorganisms to establish their niche.

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 [8–10]. 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

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

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

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 dismutation of H2O2 in microorganisms occurs mainly via three phylogenetically unrelated catalases: monofunctional catalase, catalase-peroxidase, and

Bacterial monofunctional catalases are classified into clades 1–3 according to phylogenetic analysis based on their amino acid sequences [14, 15]. Clade 1

Mn-catalase [2, 13]. Here, we focus on monofunctional catalases.

**504**

within biofilm.

essential.

the environment.

**2. Phylogeny 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 [21–24]. 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 [26–29]. 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 (*k*cat/*K*m) 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* 

**507**

well conserved in PKTA.

in EKTA.

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

*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

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 *V*max and *K*m of 8.0 × 105 μmol H2O2/μmol heme/s and 35 mM for H2O2, respectively, as determined spectrophotometrically. The

reported clade 3 catalases owing to the low *K*m value [31]. Additionally, because

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

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 *V*max and *K*m of 1.5 × 106 μmol H2O2/μmol heme/s and 40 mM for H2O2, respectively, as determined by spectropho-

highest among reported clade 1 catalases owing to the high *k*cat and low *K*m 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

The catalase from *P*. *piscatorii* T-3 (PKTA) can be purified by one step of anionchromatography and one step of hydrophobic chromatography [35]. The purified PKTA exhibits an activity of 222,000 U/mg protein under standard reaction conditions, with *V*max and *K*m of 2.4 × 105 μmol H2O2/μmol heme/s and 75 mM for H2O2,

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

(VKTA) can be purified by two steps of

cells, high affinity to H2O2 and high catalytic

/s/M, which is the highest among

/s/M, which is the

/s/M as determined

*DOI: http://dx.doi.org/10.5772/intechopen.95489*

and/or environmental selection processes. The catalase from *V*. *rumoiensis* S-1T

catalytic efficiency *k*cat/*K*m of VKTA is 2.3 × 107

this amino acid substitution on the function is unknown.

tometry [28]. The catalytic efficiency *k*cat/*K*m of EKTA is 3.8 × 107

respectively. The catalytic efficiency *k*cat/*K*m of PKTA is 3.2 × 106

of the fragility of *V*. *rumoiensis* S-1T

*Evolutionary Strategies of Highly Functional Catalases for Adaptation to High H2O2 Environments DOI: http://dx.doi.org/10.5772/intechopen.95489*

*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 *V*max and *K*m of 8.0 × 105 μmol H2O2/μmol heme/s and 35 mM for H2O2, respectively, as determined spectrophotometrically. The catalytic efficiency *k*cat/*K*m of VKTA is 2.3 × 107 /s/M, which is the highest among reported clade 3 catalases owing to the low *K*m 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.

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 *V*max and *K*m of 1.5 × 106 μmol H2O2/μmol heme/s and 40 mM for H2O2, respectively, as determined by spectrophotometry [28]. The catalytic efficiency *k*cat/*K*m of EKTA is 3.8 × 107 /s/M, which is the highest among reported clade 1 catalases owing to the high *k*cat and low *K*m 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 anionchromatography and one step of hydrophobic chromatography [35]. The purified PKTA exhibits an activity of 222,000 U/mg protein under standard reaction conditions, with *V*max and *K*m of 2.4 × 105 μmol H2O2/μmol heme/s and 75 mM for H2O2, respectively. The catalytic efficiency *k*cat/*K*m 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.

*Antioxidants - Benefits, Sources, Mechanisms of Action*

from marine environments or organisms.

genus *Vibrio* and its related genus *Aliivibrio*, strain S-1T

was identified as a new species, *E. oxidotolerans* T-2-2T

S-1T

Strain T-2-2T

purified [32]. Strain T-3-2T

purified [32].

range of strain T-3-2T

clarified. However, strain T-3-2T

been purified from *V*. *rumoiensis* S-1T

was identified as a new species, *P*. *piscatorii* T-3-2T

the periplasmic space and on the cell surface [21–24]. Therefore, *V*. *rumoiensis*

*V*. *rumoiensis* cells may help to protect the cell in high H2O2 environments. According to several reports on symbiotic or pathogenic strains involving the

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

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 [26–29]. 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,

nated 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

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

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

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

Catalases derived from H2O2-tolerant microorganisms in clade 3 and clade 1 have

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* 

. The kinetic parameters (*k*cat/*K*m) of these catalases were higher or equivalent

**5. Characteristics of catalases from H2O2-resistant bacteria**

, an H2O2-tolerant microorganism, was isolated from the upstream

is 0–30°C, and the localisation of catalase has not yet been

exhibits high resistance against H2O2. The isolate

, *P*. *piscatorii* T-3, and from *E*. *oxidotolerans*

and cell extracts of strain T-3-2T

including marine environments [30, 31]. Therefore, strain T-2-2T

cells exhibit catalase activity, and expression of catalase on the surface of

, an H2O2-tolerant microorganism, was isolated from the upstream

was predicted to be derived

.

may have origi-

and

. The growth temperature

**506**

T-2-2T

#### *Antioxidants - Benefits, Sources, Mechanisms of Action*


#### **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.*

## **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

**509**

**Figure 2.**

*substituted to "T" in PKTA. <sup>b</sup>*

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

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

[38, 39]. Therefore, the size of the key residue M167 in EKTA is the

, whereas that of M

*This amino acid residue is* 

*DOI: http://dx.doi.org/10.5772/intechopen.95489*

(Met) is 167.7 Å3

**catalase**

position of M167 in EKTA. The volume of W (Phe) is 231.7 Å3

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** 

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,

*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.* 

*This amino acid residue is modified as S-dioxymethionine.*

*The amino acid residues of narrow main channels in VSC are the same as VKTA. a*

*Evolutionary Strategies of Highly Functional Catalases for Adaptation to High H2O2 Environments DOI: http://dx.doi.org/10.5772/intechopen.95489*

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,

#### **Figure 2.**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

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

*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* 

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

Catalase is known to have high activity owing to its superior substrate selectivity

**508**

**channel**

*marked by underlined text.*

**Figure 1.**

*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. a This amino acid residue is substituted to "T" in PKTA. <sup>b</sup> This amino acid residue is modified as S-dioxymethionine.*

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.

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


**511**

**Figure 3.**

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

Clade 1 3 3

*The ratio of compound I formation rate using peracetic acid (a) to catalase activity using H2O2 (b). <sup>b</sup>*

*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].*

**EKTA PKTA VKTA**

**Yes Yes Yes**

**28,000 20,000** 7,300

*DOI: http://dx.doi.org/10.5772/intechopen.95489*

**Phylogeny**

position

(U/mg)c

(U/mg)d

*a*

*c*

*d*

**Table 1.**

Extended of phylogenetic

Purified catalase activity

Catalase activity of cell extract

*Determined by spectrophotometry.*

*Determined by oxygen electrode analysis.*

*Standard reaction conditions of 30 mM H2O2 at pH 7.*

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

*Evolutionary Strategies of Highly Functional Catalases for Adaptation to High H2O2 Environments DOI: http://dx.doi.org/10.5772/intechopen.95489*


*c Determined by oxygen electrode analysis.*

*d Standard reaction conditions of 30 mM H2O2 at pH 7.*

#### **Table 1.**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

**structure of catalases**

**Bottle neck structure**

**Enzymatic feature**

**Cellular features**

extract

Kinetic parameters for H2O2

Percentage of catalase in cell

**Location of isolation** Upstream of the

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

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** 

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

BN2–BN4 L149, **M167**, I180 V158, **W176**, T189 V154, **W172**, V185 The size of BN2–BN4 164.6, **167.7**, 164.9 150.6, **231.7**, 120.0 150.6, **231.7**, 139.1

b/a ratioa **1.4 0.0056** ND

*V*max (/s) 1.5 × 106 <sup>b</sup> 2.4 × 105 <sup>c</sup> 8.0 × 105 <sup>b</sup> *K*m (mM) 40b 75c 35b *k*cat/*K*m (/M/s) 3.8 × 107 <sup>b</sup> 3.2 × 106 <sup>c</sup> 2.3 × 107 <sup>b</sup>

> drain (**6–38 mM H2O2**)

**Involved bacteria** Gram positive Gram negative Gram negative

**EKTA PKTA VKTA**

**6.5% 10%** 1.8%

Upstream of the drain (**6–38 mM H2O2**)

Downstream of the drain (1.5–6 mM H2O2)

catalytic reactions in the presence of high concentrations of H2O2.

**510**

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

#### **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].*

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.

## **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.

**513**

**Author details**

Isao Yumoto1,2\*, Yoshiko Hanaoka1,2 and Isao Hara1,3

2 Graduate School of Agriculture, Hokkaido University, Japan

Catalase website (http://www.catalase.com/index.htm)

(http://pfam.xfam.org/family/PF00199#tabview=tab3) Catalase (enzyme nomenclature designation [EC] 1.11.1.6)

Science and Technology (AIST), Sapporo, Japan

\*Address all correspondence to: i.yumoto@aist.go.jp

provided the original work is properly cited.

3 Simadzu Co. Ltd., Kyoto, Japan

1 Bioproduction Research Institute, National Institute of Advanced Industrial

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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

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

Takebe, and Mr. Hideyuki Kimoto for providing technical assistance.

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.

*DOI: http://dx.doi.org/10.5772/intechopen.95489*

**Acknowledgements**

**Conflict of interest**

**Appendices and nomenclature**

Website resources:

EMBEL-EBI Catalase

interest.

*Evolutionary Strategies of Highly Functional Catalases for Adaptation to High H2O2 Environments DOI: http://dx.doi.org/10.5772/intechopen.95489*

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.

## **Acknowledgements**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

(8°C, 6–38 mM H2O2), *E*. *oxidotolerans* T-2-2T

to the intrinsic genomic architectural dynamics of these taxa.

**10. Conclusion and future studies**

*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 environ-

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.

ments containing high H2O2 by genomic altering specific characteristics.

**bacteria**

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.

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

Results of a screening of bacterial strains adapted to high H2O2 environments

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

and *P*. *piscatorii* T-3T

and T-3-2

**512**

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 (http://www.catalase.com/index.htm) EMBEL-EBI Catalase (http://pfam.xfam.org/family/PF00199#tabview=tab3) Catalase (enzyme nomenclature designation [EC] 1.11.1.6)

## **Author details**

Isao Yumoto1,2\*, Yoshiko Hanaoka1,2 and Isao Hara1,3

1 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan

2 Graduate School of Agriculture, Hokkaido University, Japan

3 Simadzu Co. Ltd., Kyoto, Japan

\*Address all correspondence to: i.yumoto@aist.go.jp

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Yoshimune K; Heme content of

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abs/pii/S0969212600000654

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Stackebrandt E; *Psychrobacter* 

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10.1099/ijs.0.000149.

Press; 2013. p 137-158.

reflected by

article/pii/S1389172308700045

Hara I, Matsuyama H, Yumoto I; Growth-dependent catalase localization in *Exiguobacterium* 

*oxidotolerans* T-2-2T

pone.0076862

T-2-2T

**516**

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**519**

**Chapter 25**

**Abstract**

important antioxidant potential.

**1. Introduction**

Antioxidant Properties

(Southern, Tunisia)

of Metabolites from New

*Sana Gammoudi, Ines Dahmen-Ben Moussa,* 

Extremophiles Microalgal Strain

*Neila Annabi-Trabelsi, Habib Ayadi and Wassim Guermazi*

With the demand for bioproducts that can provide benefits for biotechnology sectors like pharmaceuticals, nutraceuticals, and cosmeceuticals, the exploration of microalgal products has turned toward extremophiles. This chapter is intended to provide an insight to most important molecules from halotolerant species, the cyanobacteria *Phormidium versicolor* NCC-466 and *Dunaliella* sp. CTM20028 isolated from Sfax Solar Saltern (Sfax) and Chott El-Djerid (Tozeur), Tunisia. These microalgae have been cultured in standard medium with a salinity of 80 PSU. The *in vitro* antioxidant activities demonstrated that extremolyte from *Dunaliella* and *Phormidium* as, phycocaynin, lipids, and polyphenol compound presents an

The primary producers of oxygen in aquatic environments are algae, especially

recycling through photosynthesis [1]. Microalgae have been divided into ten groups, which refer to the color of the cell including: Cyanobacteria, blue-green algae; Chlorophyta, green algae; Rhodophyta, red algae; Glaucophyta; Euglenophyta; Haptophyta; Cryptophyta; photosynthetic Stramenopiles; Dinophyta; and Chlorarachniophyta [2]. Cyanobacteria are much closer to bacteria in terms of structure and their cells lack both nucleus and chloroplasts. Cyanobacteria are also known as a source of pigments, chlorophyll (a), phycocyanin, phycoerythrin, xanthophyll, and ß-carotene. Microalgae are widely distributed in nature and adapted to different environments from fresh to hypersaline water ecosystems. Salt lakes in arid regions (sabkhas) and solar salterns are an examples of high salty environments inhabited by extremely halophilic microorganisms that include halophilic Archaea (halobacteria), halophilic cyanobacteria, and green algae [3–5]. These microorganisms must have specific adaptive strategies for surviving in high salinity conditions to prevent the loss of cellular water under high osmolarity in

planktonic microalgae. They play an important role in carbon dioxide (CO2)

**Keywords:** microalgae, halophile, biomolecule, antioxidant properties

## **Chapter 25**
