**1. Introduction**

Oceans cover 71% of the earth's surface and are home to a diverse range of species, such as algae, bacteria, fungi, sponges, and fish. The sea is a challenging place to inhabit because it has both deep and shallow areas with different temperatures and pressures, salinity changes, different pHs, light, hydrostatic pressure, and the distribution of different nutrients, all of which lead to a wide variety of marine organisms with unique characteristics [1–3].

Marine microorganisms are a promising source for discovering novel enzymes because of their distinctive natural environments, physiological traits, distinctive metabolic processes, and use of varied nutrients [4,5]. Marine bacteria are a diverse group of marine microorganisms that have developed physiological adaptations in response to various environmental factors and evolutionary processes. They also produce a variety of hydrolyzing enzymes, such as amylases, lipases, and proteases, which may have applications in contemporary biotechnology [6]. The main benefits of employing microorganisms for enzyme synthesis over plants and animals are rapid growth, huge production capacity, and simple enzyme extraction from bacteria [5,6].

The buoyancy of fish in water causes bacteria to cover the outer surface of their bodies, and as a result, fish are in continuous contact with the microorganisms that cover their bodies. Some of these microorganisms do not live on the surface of the fish but are instead

**Citation:** Erfanimoghadam, M.R.; Homaei, A. Identification of New Amylolytic Enzymes from Marine Symbiotic Bacteria of *Bacillus* Species. *Catalysts* **2023**, *13*, 183. https:// doi.org/10.3390/catal13010183

Academic Editors: Zhilong Wang and Tao Pan

Received: 4 December 2022 Revised: 6 January 2023 Accepted: 11 January 2023 Published: 13 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

found as part of the microbes that live within the fish body, such as the oral cavity [7]. The combination of all environmental factors for fish symbiotic bacteria results in the development of enzymes with exceptional features of the bacteria in this milieu [8,9].

Amylases are one of the most important sources of industrial enzymes with many applications in various industries, such as pharmaceuticals, food industries, auxiliary food preparation, detergents, papermaking, and textiles. They provide the hydrolysis of starch into small sugar units of dextrin or smaller glucose polymers and are classified into three groups, namely α-amylases, β-amylases, and γ-amylases. α-amylases cleave the bond (α (1→4)) between adjacent glucose units in the linear chain of amylose in starch carbohydrates [10]. Since α-amylase has multiple cleavage sites, α-amylase is faster than β-amylase. In addition, α-amylase enzymes have the necessary stability against high temperatures, especially α-amylase synthesized from *Bacillus subtilis* and *Archaea* bacteria species, which are resistant to heat [11]. Various physical and chemical factors, such as temperature, pH, incubation period, carbon sources as inducers, surfactants, nitrogen sources, phosphate, and different metal ions, affect the production of α-amylase [12].

Although the production of α-amylase enzyme by Gram-negative bacterial strains such as *B.* sp., *Aeromonas* sp., and *Stenotrophomonas* sp. has been reported, the production of α-amylase isolated from fish intestinal bacteria is still ambiguous and has not been widely studied [13]. So far, no research has been performed on the isolation of α-amylase enzymeproducing bacteria coexisting with the digestive systems of *Sillago sihamas* and *Rastrelliger Kanagurta* fish from the southern coasts of Iran. In this study, the symbiotic bacteria of the intestine of *Sillago sihamas* and *Rastrelliger Kanagurta* fish were isolated, and then α-amylase enzyme-producing bacteria were screened, and those with the highest potential to produce α-amylase were identified by biochemical, morphological, and molecular methods, and finally, some biochemical characteristics of the enzyme were investigated.

## **2. Results and Discussion**

#### *2.1. Primary and Secondary Screening of α-Amylase-Producing Bacteria*

In this study, 22 colonies were isolated from the collected samples of *Rastrelliger kanagurta* and *Sillago sihama*, of which 12 colonies were isolated from the intestine of *Sillago sihama* and 10 colonies were isolated from the intestine of *Rastrelliger kanagurta*. Among the twenty-two isolated bacterial strains, ten bacterial strains were able to grow in a special culture medium containing starch, five bacterial strains were able to produce strong halos by Lugol's test, and other bacterial strains had a weak or moderate halo creation ability by Lugol's test (Figure 1). The results revealed that these bacterial strains produce the α-amylase enzyme. In this experiment, starch was employed as a carbon source, and only those bacteria that had α-amylase enzyme were able to hydrolyze starch in this culture medium and grow in this specific medium (Tables 1 and 2). For secondary screening, among the bacteria isolated from the intestines of *Sillago sihama* and *Rastrelliger kanagurta*, five bacterial strains with the highest degrees of halo formation (strong and medium) were chosen in the primary screening stage to measure enzyme activity. Meanwhile, four strains of *B. subtilis* strain HFBP08, *B. subtilis* strain ZIM3, *B. subtilis* strain SXK, and B. subtilis strain soil G2B had the highest amount of enzyme activity with the activity levels of 0.074, 0.048, 0.061, and 0.072, respectively. Finally, these four potential α-amylase enzyme-producing strains were selected for further analyses and molecular identification. The results indicated that *Rastrelliger kanagurta* and *Sillago sihama* fish species have rich sources of α-amylaseproducing bacteria. The most potent bacterium among those producing enzymes was *B. subtilis*. In general, based on our findings, this bacterial strain can be a suitable source for the production of heat-tolerant amylase enzymes with high functional stability for use in industry. For example, α-amylase generated by these *Bacillus* species is used in the confectionery industry because of its temperature resistance, as well as starch liquefaction, processing, and application. They have a wide range of commercial applications and are especially useful in the food and pastry industries. Earlier research, similar to the current

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 3 of 11

study, used a specialized growth medium containing starch for the first screening of bacteria that produce α-amylase. [14,15]. similar to the current study, used a specialized growth medium containing starch for the first screening of bacteria that produce α-amylase. [14,15].

applications and are especially useful in the food and pastry industries. Earlier research,

**Figure 1.** Pure culture of isolated bacteria in nutrient agar medium (**a**), growth of α-amylase-producing bacteria in a specific solid culture medium containing starch (**b**), and a clear halo around the colony of α-amylase-producing bacteria using Lugol's solution (**c**). **Figure 1.** Pure culture of isolated bacteria in nutrient agar medium (**a**), growth of α-amylaseproducing bacteria in a specific solid culture medium containing starch (**b**), and a clear halo around the colony of α-amylase-producing bacteria using Lugol's solution (**c**).

**Table 1.** Evaluation of bacterial strains capable of producing α-amylase from the intestines of *Sillago sihama.*  **Table 1.** Evaluation of bacterial strains capable of producing α-amylase from the intestines of *Sillago sihama*.



**Table 2.** Assessment of bacterial strains capable of generating α-amylase from the intestines of *Rastrelliger kanagurta*.

#### *2.2. Molecular Identification of Potential α-Amylase-Producing Bacterial Strains*

Identification techniques based on molecular studies are essential and accurate instruments for the proper characterization of microbial species. In this regard, four bacterial strains (HR13, HR16, HR15, and HR14) that had the highest α-amylase activity were identified by 16S rRNA gene analysis (Figure 2). All four strains with the capacity to produce α-amylase enzyme aligned most closely to the *Bacillus* genus, Bacillaceae family, and *Bacillus subtilis* species, according to the NCBI database's analysis of the nucleotide sequence of the 16S rRNA gene of isolated bacteria. Based on these results, the bacterial strains (HR13, HR16, HR15, and HR14) have the highest similarity (99%) with *B. subtilis* strains HFBP08, ZIM3, SXK, and soilG2B. According to the results of 16S rRNA gene sequences, *B. subtilis* strain HR13, *B. subtilis* strain HR14, *B. subtilis* strain HR15 B., and *B. subtilis* strain HR16 were registered in the NCBI database with accession numbers MZ571841, MZ571838, MZ571839, and MZ571840, respectively. The phylogenetic tree of *B. subtilis* strains HR13, HR14, HR15 B., and HR16 isolated from the fish intestine in this study, as well as sequences available in NCBI for *B. Methanobacterium formicicum*, *B. infantis* strain C4, *B. amyloliquefaciens* strain TS.18 S.BK, *B. velezensis* strain CB02999, *B. tequilensis* strain HYM43, and *B. mojavensis* strain WSE-KSU305, were drawn and their evolutionary relationships were investigated (Figure 3). The evolutionary relationships of the strains obtained in this research with other *Bacillus* species, including *B. infantis*, *B. amyloliquefaciens*, *B. velezensis*, *B. tequilensis*, and *B. mojavensis*, are shown in a phylogenetic tree. Therefore, the present study shows that the strains belonging to *B. subtilis* are potential bacteria for the production of the α-amylase enzyme.

In 2014, Castro et al. isolated endophytic microorganisms symbiotic with leaves and branches of mangrove trees in Brazil from two mangrove species, namely *Rhizophora mangle* and *Avicennianitida. Bacillus* was the most isolated genus from mangrove trees in this region [16]. Similar to the present study, bacteria belonging to the *Bacillus* genus, including *B. subtilis*, *B. stearothermophilus*, and *B. amyloliquefaciens*, have been identified as potential α-amylase enzyme-producing bacteria [17–19]. *B. amyloliquefaciens* produces the most α-amylase enzyme in the world, and the α-amylase generated by this bacterium was the first enzyme employed in the industry for starch sweetening and liquefaction. [20–22]. Therefore, these findings demonstrate that *bacilli* have the unique capacity to synthesize α-amylase enzyme.

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 5 of 11

weight of 1500 bp; Lad is a molecular marker.

**Figure 2.** Identification of 16S rRNA gene bands of enzyme-producing bacteria with a molecular **Figure 2.** Identification of 16S rRNA gene bands of enzyme-producing bacteria with a molecular weight of 1500 bp; Lad is a molecular marker.

**Figure 2.** Identification of 16S rRNA gene bands of enzyme-producing bacteria with a molecular

**Figure 3.** Phylogenetic tree of 16S rRNA nucleotide sequences in the analyzed strains (0.05 nucleotide replacement rate per site). **Figure 3.** Phylogenetic tree of 16S rRNA nucleotide sequences in the analyzed strains (0.05 nucleotide replacement rate per site).

**Figure 3.** Phylogenetic tree of 16S rRNA nucleotide sequences in the analyzed strains (0.05 nucleo-*2.3. The Effect of Temperature and pH on the Activity and Stability of the α-Amylase Enzyme*

region [16]. Similar to the present study, bacteria belonging to the *Bacillus* genus, including

tide replacement rate per site). In 2014, Castro et al. isolated endophytic microorganisms symbiotic with leaves and branches of mangrove trees in Brazil from two mangrove species, namely *Rhizophora mangle* and *Avicennianitida. Bacillus* was the most isolated genus from mangrove trees in this In 2014, Castro et al. isolated endophytic microorganisms symbiotic with leaves and branches of mangrove trees in Brazil from two mangrove species, namely *Rhizophora mangle* and *Avicennianitida. Bacillus* was the most isolated genus from mangrove trees in this region [16]. Similar to the present study, bacteria belonging to the *Bacillus* genus, including The effect of temperature on α-amylase enzyme activity in *B. subtilis* strains HR13 and HR16 showed that both strains display maximum activity at 60 ◦C (Figure 4). At temperatures higher than 80 ◦C, the enzyme activity of both strains suddenly drops, so that at a temperature of 90 ◦C, the enzyme activity of the HR16 strain reaches almost zero.

α-amylase enzyme.

**Figure 4.** The effect of temperature on the enzyme activity levels of HR13 (■) and HR16 (▲) strains; **Figure 4.** The effect of temperature on the enzyme activity levels of HR13 () and HR16 (N) strains; the activity at the optimal temperature was taken as 100%.

*B. subtilis*, *B. stearothermophilus*, and *B. amyloliquefaciens*, have been identified as potential α-amylase enzyme-producing bacteria [17–19]. *B. amyloliquefaciens* produces the most αamylase enzyme in the world, and the α-amylase generated by this bacterium was the first enzyme employed in the industry for starch sweetening and liquefaction. [20–22]. Therefore, these findings demonstrate that *bacilli* have the unique capacity to synthesize

*2.3. The Effect of Temperature and pH on the Activity and Stability of the α-Amylase Enzyme* 

The effect of temperature on α-amylase enzyme activity in *B. subtilis* strains HR13 and HR16 showed that both strains display maximum activity at 60 °C (Figure 4). At temperatures higher than 80 °C, the enzyme activity of both strains suddenly drops, so that at a temperature of 90 °C, the enzyme activity of the HR16 strain reaches almost zero.

the activity at the optimal temperature was taken as 100%. The effect of different pH values on the enzyme activity of two strains, *B. subtilis* HR13 and *B. subtilis* HR16, shows their maximum enzyme activity at pH 5 and 7, respectively (Figure 5). The α-amylase enzyme activity of HR16 had a sharp drop after the optimum pH so that at pH values of 8 and 9, 35% and 28% of the initial activity of the enzyme was retained, respectively. In the case of the HR13 strain, the activity of the enzyme did The effect of different pH values on the enzyme activity of two strains, *B. subtilis* HR13 and *B. subtilis* HR16, shows their maximum enzyme activity at pH 5 and 7, respectively (Figure 5). The α-amylase enzyme activity of HR16 had a sharp drop after the optimum pH so that at pH values of 8 and 9, 35% and 28% of the initial activity of the enzyme was retained, respectively. In the case of the HR13 strain, the activity of the enzyme did not significantly decrease after reaching the optimal pH, and the enzyme maintained more than 75% of its initial activity in the range of pH 6–8.

not significantly decrease after reaching the optimal pH, and the enzyme maintained more than 75% of its initial activity in the range of pH 6–8. Irreversible thermal inactivation of enzymes isolated from *B. subtilis* strains HR13 and HR16 at temperatures of 80 and 90 °C demonstrated that at 80 °C with increasing incubation time (Figure 6), the amount of enzyme activity decreased, and after temperature incubation for up to 10 min, the enzyme activity in the HR13 B. *subtilis* strain reaches less than half. However, the enzyme of *B. subtilis* strain HR16 maintained > 50% of its initial activity after a temperature incubation for 20 min at 80 °C. Notably, after 60 min of Irreversible thermal inactivation of enzymes isolated from *B. subtilis* strains HR13 and HR16 at temperatures of 80 and 90 ◦C demonstrated that at 80 ◦C with increasing incubation time (Figure 6), the amount of enzyme activity decreased, and after temperature incubation for up to 10 min, the enzyme activity in the HR13 B. *subtilis* strain reaches less than half. However, the enzyme of *B. subtilis* strain HR16 maintained > 50% of its initial activity after a temperature incubation for 20 min at 80 ◦C. Notably, after 60 min of temperature incubation, the activity of both enzyme strains reached almost zero. *Catalysts* **2023**, *13*, x FOR PEER REVIEW 7 of 11

**Figure 5.** The impact of pH on the enzyme activity levels of HR13 () and HR16 (N); the activity at the optimal pH was taken as 100%.

**Figure 5.** The impact of pH on the enzyme activity levels of HR13 (■) and HR16 (▲); the activity at

the optimal pH was taken as 100%.

the optimal pH was taken as 100%.

0

20

40

60

**Relative activity (%)**

80

100

**Figure 5.** The impact of pH on the enzyme activity levels of HR13 (■) and HR16 (▲); the activity at

 HR13 HR16

3 5 7 9 11 13

**pH**

**Figure 6.** Thermal stability of the α-amylase enzyme activity of HR13 () and HR16 (N) after incubating the enzymes in a water bath at 80 ◦C (**a**) and 90 ◦C (**b**) for different time periods.

At 90 ◦C, irreversible thermal inactivation of the enzyme decreases enzyme activity substantially faster than at 80 ◦C. After 30 min of temperature incubation, the enzyme activity of *B. subtilis* strain HR13 reaches less than 20% of its initial activity. About 22% of the enzyme's initial activity was still present in the *B. subtilis* strain HR16 enzyme after 30 min of incubation at 90 ◦C. The irreversible inactivation of enzymes isolated from HR13 and HR16 was investigated and compared at an alkaline pH of 8 (Figure 7). Both forms of the enzyme exhibit a notable decline in activity at pH 8 with extended incubation times. The enzymes of strains HR13 and HR16 preserved 56% and 63% of their initial activity after 30 min of incubation at pH 8, respectively. At pH 12, the activity of the free enzyme decreased as the incubation time increased. After 60 min of incubation at pH 8, the free enzyme in both enzyme forms was reversible to about 18% of its initial activity.

**Figure 7.** Irreversible inactivation of the α-amylase enzyme in HR13 () and HR16 (N) after different time periods of incubation at pH 8.

**Figure 6.** Thermal stability of the α-amylase enzyme activity of HR13 (■) and HR16 (▲) after incu-

At 90 °C, irreversible thermal inactivation of the enzyme decreases enzyme activity substantially faster than at 80 °C. After 30 min of temperature incubation, the enzyme activity of *B. subtilis* strain HR13 reaches less than 20% of its initial activity. About 22% of the enzyme's initial activity was still present in the *B. subtilis* strain HR16 enzyme after 30 min of incubation at 90 °C. The irreversible inactivation of enzymes isolated from HR13 and HR16 was investigated and compared at an alkaline pH of 8 (Figure 7). Both forms of the enzyme exhibit a notable decline in activity at pH 8 with extended incubation times. The enzymes of strains HR13 and HR16 preserved 56% and 63% of their initial activity after 30 min of incubation at pH 8, respectively. At pH 12, the activity of the free enzyme decreased as the incubation time increased. After 60 min of incubation at pH 8, the free

bating the enzymes in a water bath at 80 °C (**a**) and 90 °C (**b**) for different time periods.

enzyme in both enzyme forms was reversible to about 18% of its initial activity.

#### **Figure 7.** Irreversible inactivation of the α-amylase enzyme in HR13 (■) and HR16 (▲) after differ-**3. Materials and Methods**

ent time periods of incubation at pH 8. *3.1. Materials Used for the Collection of Short Fish Samples*

**3. Materials and Methods**  All reagents were purchased from Merck Co. (Darmstadt, Germany).

#### *3.1. Materials Used for the Collection of Short Fish Samples 3.2. Collection of Sillago sihama and Rastrelliger kanagurta Fish*

All reagents were purchased from Merck Co. (Darmstadt, Germany). *3.2. Collection of Sillago sihama and Rastrelliger kanagurta Fish*  Freshly caught fish samples of *Sillago sihama* and *Rastrelliger kanagurta* were purchased from the Qeshm fishmongers' market and transferred to the laboratory in a flask containing ice at a temperature of 4–10 ◦C.

#### Freshly caught fish samples of *Sillago sihama* and *Rastrelliger kanagurta* were purchased from the Qeshm fishmongers' market and transferred to the laboratory in a flask *3.3. Isolation of Intestinal Bacteria from Sillago sihama and Rastrelliger kanagurta Fish*

containing ice at a temperature of 4–10 °C. *3.3. Isolation of Intestinal Bacteria from Sillago sihama and Rastrelliger kanagurta Fish*  First, the abdominal surface of the fish was cleaned with 70% alcohol. After opening the stomach of the fish with a surgical blade, the intestines were removed under sterile conditions. After homogenizing and diluting the samples with physiological serum, they First, the abdominal surface of the fish was cleaned with 70% alcohol. After opening the stomach of the fish with a surgical blade, the intestines were removed under sterile conditions. After homogenizing and diluting the samples with physiological serum, they were cultured in nutrient agar culture medium and kept in a greenhouse at 30 ◦C for 48 h in order to isolate bacteria. After the incubation period, the plates were examined morphologically (color and appearance) under the laminar hood, and the colonies were purified.

#### were cultured in nutrient agar culture medium and kept in a greenhouse at 30 °C for 48 h *3.4. Primary Screening of α-Amylase-Producing Bacteria*

in order to isolate bacteria. After the incubation period, the plates were examined morphologically (color and appearance) under the laminar hood, and the colonies were purified. Bacteria isolated from the intestines of *Sillago sihama* and *Rastrelliger kanagurta* were cultured on a special culture medium containing starch (1%) and kept in a greenhouse at 30 ◦C for 48 h. After the greenhouse period, the growth of bacteria on the starch culture medium was analyzed, and the samples exhibiting the ability to grow in this medium were selected for further analyses. At this stage, Lugol's solution was poured onto the special culture medium containing starch; then, based on the diameter of the clear halo around the bacterial colony, which indicates starch hydrolysis and enzyme production by bacteria, the α-amylase enzyme-producing colonies were isolated and selected.
