Isolation and Characterization of a Thermaerobacillus caldiproteolyticus-like Strain Producing Extracellular Amylase from the Nelumwewa Geothermal Spring, Sri Lanka

Abstract

The growing demand for efficient sustainable biocatalysts for industrial applications has driven the exploration of extremozymes from extremophiles, particularly those thriving in geothermal environments. This study aimed to isolate and characterize extracellular amylase-producing thermophilic bacteria from the Nelumwewa geothermal spring in Sri Lanka, an underexplored ecosystem. Among the isolated thermophilic bacterial strains, NW2 isolates exhibited a prominent extracellular amylase activity. Molecular characterization by 16S rRNA gene sequencing confirmed the close phylogenetic relationship between NW2 and Thermaerobacillus caldiproteolyticus, which is well-known for thermostable proteases. Biochemical assays revealed optimal amylase activity of NW2 isolate at 60 °C and pH 8.0, with a crude enzyme activity of 0.85 U/mL. The enzyme demonstrated efficient hydrolysis of raw cassava starch, highlighting its potential for industrial applications in food, biofuel, and detergent industries. This study reports the first T. caldiproteolyticus-like strain from Sri Lanka with significant extracellular amylase activity, emphasizing the biotechnological potential of geothermal springs as sources of novel extremozymes. These findings contribute to the growing repository of thermostable enzymes, highlighting the need for further exploration of Sri Lanka’s geothermal microbial diversity for industrial biocatalysts.

1. Introduction

Many industrial processes use large amounts of expensive and hazardous synthetic chemical catalysts, which pose serious risks to the environment and human health. In response, biocatalysts are receiving greater attention as sustainable and eco-friendly alternatives with superior selectivity, efficiency, and cost-effectiveness [1,2]. Microbial extremozymes are a unique class of biocatalysts that exhibit remarkable stability and catalytic efficiency under extreme environmental conditions, such as elevated temperatures, extreme pH, and high salinity [3,4,5]. Among the various extremozymes, thermozymes produced by thermophilic bacteria are of particular interest in modern biotechnological industries due to their high thermostability, which is essential for maintaining enzymatic activity under the harsh conditions of industrial processes [6]. Thermophilic bacteria are broadly classified based on their optimal growth temperatures into moderate (optimal growth 45–80 °C) and extreme (optimal growth > 80 °C) types [7,8]. Bacterial thermozymes, such as amylases, proteases, lipases, polymerases, cellulases, and chitinases have gained significant industrial importance due to their efficient activity under extreme conditions [6].

Among microbial extremozymes, thermostable amylases are of commercial significance in various industries representing approximately 25% of the global enzyme market. These amylases hydrolyze starch at elevated temperatures, eliminating the need for energy-intensive cooling systems and contributing to substantial reductions in processing costs [9]. They offer promising operational advantages across a range of industrial sectors, including food processing, pharmaceutical, pulp and paper, textile manufacturing, detergent, biofuel production, bioremediation, and waste management [6,10,11]. Thermostable amylases are primarily produced for industrial applications by several well-characterized bacterial genera, such as Bacillus [12] and Anoxybacillus [13,14,15], due to their high enzymatic yield, remarkable thermostability, and scalability. Moreover, numerous studies have been conducted on the isolation and characterization of bacterial species that produce thermostable amylases from diverse ecological niches. Thermostable amylases have been identified in species, such as Streptomyces erumpens, Thermobifida fusca, and Bacillus strains isolated from soil environments [16,17,18,19]. In the ongoing search for excellent thermostable amylases for industrial applications, several researchers have successfully isolated and characterized thermostable amylases from thermostable bacteria that thrive in thermal spring environments [15,20,21,22,23,24].

Despite significant advancements in the identification of novel extremophiles for industrial applications, the geothermal ecosystems of Sri Lanka remain largely underexplored. A study has been reported that explored the microbial diversity of a geothermal spring in Sri Lanka [25]. However, this study did not report the presence of well-known thermostable amylase-producing bacteria. This highlights a significant gap in the understanding of Sri Lanka’s geothermal microbial diversity in the context of industrially important extremozymes. Therefore, the present study was conducted with the objective of the isolation and molecular characterization of thermophilic bacteria producing extracellular amylases from Sri Lankan geothermal springs and assessing their potential for industrial applications.

2. Materials and Methods

2.1. Water Sample Collection

Water samples were collected into sterile vacuum flasks from Nelumwewa geothermal spring (7°53′28.0″ N 81°11′58.2″ E) in Polonnaruwa district of Sri Lanka (Figure 1). Water temperature and pH of the hot water spring were recorded. The water samples were immediately transferred to the laboratory for thermophilic bacterial isolation.

2.2. Isolation of Thermophilic Bacteria from Water Samples

An aliquot of 2 mL from each water sample was inoculated separately into 100 mL of nutrient broth (NB) (Himedia, Thane, India) and incubated overnight at 55 °C with shaking at 150 rpm for enrichment. Then, the incubated samples were serially diluted, and 100 µL from each sample was spread on individual nutrient agar plates. The bacterial colonies with different morphology were carefully selected, and pure cultures were isolated on nutrient agar plates, and incubated at 55 °C overnight. The pure cultures were prepared by streaking and subculturing and maintained on nutrient agar plates at 4 °C. For long-term storage, 20% glycerol stocks were prepared from each pure culture and stored at −80 °C. Gram’s staining was conducted to differentiate Gram-positive and Gram-negative bacterial isolates.

2.3. Catalase Test

To identify the catalase-producing bacteria, a small amount of each bacterial isolate was placed on individual clean glass slides. A drop of 3% hydrogen peroxide solution was added to each bacterial sample, and formation of bubbles was observed. The bacterial isolates that produced bubbles were identified to be catalase-producing bacteria [27].

2.4. Screening of Thermophilic Bacterial Isolates for Extracellular Amylase Production

Extracellular amylase-producing bacterial isolates were screened by growing pure isolates on starch agar plates (peptone 5 g/L, NaCl 5 g/L, yeast extract 1.5 g/L, beef extract 1.5 g/L, soluble starch 2 g/L, agar 15 g/L, pH 7.5) (Himedia, India) at 55 °C overnight. Then, the plates were flooded with iodine solution (2% I₂ and 0.2% KI) (Himedia, India) for 5 min, and iodine solution was decanted. The isolates that gave a clear zone around the colonies were selected and sub-cultured to maintain pure cultures, and they were identified as extracellular amylase-producing thermophilic bacteria [28].

2.5. Screening of Thermophilic Bacterial Isolates for Extracellular Protease Production

All the bacterial isolates were screened for extracellular protease by growing pure isolates on skim milk agar plates (5.0 g/L peptone, 3.0 g/L yeast extract, 1.0 g/L skim milk powder, pH 7.5) (Himedia, India) at 55 °C overnight. The thermophilic bacterial isolates that produced extracellular protease were identified by observing the clear zone around the bacterial colonies due to the proteolytic activity.

2.6. Molecular Characterization of Isolated Thermophilic Bacteria by 16S rRNA Gene Sequencing and Phylogenetic Analysis

For 16S rRNA gene bidirectional sequencing with the 27F (5′-AGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) universal primer pair, all the six pure isolates were sent to Macrogen, Inc., Seoul, Republic of Korea through Genetech Sri Lanka Pvt. Ltd., Colombo, Sri Lanka. The consensus sequences were generated using BioEdit version 7. To identify the thermophilic bacterial isolates, all the sequences were blasted against NCBI rRNA/ITS databases available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 5 May 2025). The partial 16S rRNA gene sequences of isolated thermophilic bacteria were deposited in NCBI GenBank under the accession numbers PV489066, PV489057, and PV489038. Phylogenetic analysis was conducted using MEGA software version 12 [29]. The phylogenetic tree was constructed using the neighbor-joining method with bootstrap value of 1000, and the tree was visualized with modifications using tvBOT [30].

2.7. Determination of the Optimum Growth Temperature for the NW2 Isolate

The NW2 isolate was cultured in 20 mL of NB (beef extract 3 g/L, peptone 5 g/L, and NaCl 5 g/L, pH 7.5) at 55 °C by shaking at 150 rpm in a shaking incubator (Lab Companion, JEIO TECH, Daejeon, Republic of Korea) until the OD₆₀₀ value reached 0.5. Then, 1 mL of culture was inoculated into 60 mL of NB and incubated at different temperatures (40 °C, 45 °C, 50 °C, 55 °C, 60 °C, and 65 °C) by shaking at 150 rpm. At one-hour intervals, OD₆₀₀ values of each culture were measured using a spectrophotometer (UV-2600i Plus/2700i Plus, SHIMADZU, Kyoto, Japan), and growth curves were plotted. The exponential phase of each growth curve was selected, and corresponding OD₆₀₀ values were converted to natural log values, and growth rates were determined by linear regression analysis using Microsoft Excel^® version 2019. Each experiment was performed in triplicate.

2.8. Isolation of Crude Extracellular Amylase Enzyme from the NW2 Isolate

The crude extracellular amylase enzyme was produced and isolated from the overnight culture of NW2 strain incubated at 55 °C in 50 mL of NB (peptone 5 g/L, NaCl 5 g/L, yeast extract 1.5 g/L, beef extract 1.5 g/L, soluble starch 2 g/L, pH 7.5), with shaking at 150 rpm. Then, the culture was centrifuged at 10,000 rpm for 10 min at 4 °C using a centrifuge (Hettich, Tuttlingen, Germany). The supernatant was filtered through a bacterial filter (0.22 μm) and stored at 4 °C for conducting amylase activity assay using 3,5-Dinitrosalicylic acid (DNSA) (Sigma Aldrich, Hamburg, Germany) colorimetric assay [31].

2.9. Extracellular Amylase Activity Analysis by DNSA Assay

The extracellular amylase activity was analyzed using DNSA colorimetric assay as described previously [23,31] with slight modifications. Briefly, 0.1 mL of crude enzyme was mixed with 0.9 mL of 1% (w/v) soluble starch solution dissolved in 0.05 M phosphate buffer (pH 8) (Himedia, India) and incubated at 60 °C for 20 min. Then, 1 mL of DNSA reagent was added, and the mixture was heated in a boiling water bath for 5 min. After cooling the reaction mixture at room temperature, 8 mL of distilled water was added to the mixture, and absorbance was measured at 540 nm using a spectrophotometer. A solution without crude enzyme was used as the control. The amount of maltose (µmol) produced was determined from a maltose standard curve. All experiments were conducted in triplicate, and results are expressed as mean ± SD. One unit (U) of crude amylase activity was defined as the amount of crude enzyme that liberates 1 µmol of reducing sugars (as maltose) per minute under assay conditions, and crude extracellular amylase enzyme activity was expressed as U/mL. The crude extracellular amylase activity was calculated using the following formula [32]:

$$\mathrm{Amylase} \mathrm{activity} \left(\mathrm{U}/\mathrm{m}\mathrm{L}\right)={\displaystyle \frac{\mathrm{Maltose} \mathrm{amount} \left(\mu \mathrm{m}\mathrm{o}\mathrm{l}\right)}{\mathrm{Reaction} \mathrm{time} \left(\min \right) \times \mathrm{Volume} \mathrm{of} \mathrm{crude} \mathrm{enzyme} \left(\mathrm{mL}\right)}}$$

(1)

2.10. Determination of the Optimum Temperature for the Extracellular Amylase Activity

The optimum temperature for crude extracellular amylase activity was determined by reacting 0.1 mL of crude enzyme with 0.9 mL of 1% soluble starch solution dissolved in 0.05 M phosphate buffer (pH 7.5) for 20 min at different temperatures (40 °C, 45 °C, 50 °C, 55 °C, 60 °C, and 65 °C). Then, the activity was analyzed by DNSA assay as described above. All experiments were conducted in triplicate, and the results are expressed as mean ± SD.

2.11. Determination of Optimum pH for the Extracellular Amylase Activity

For determining optimum pH for crude extracellular amylase activity, 0.05 M sodium phosphate buffer (pH 5–8) and 0.05 M Tris-HCl buffer (pH 7–9) were used. An amount of 0.1 mL of crude enzyme was mixed with 0.9 mL of 1% soluble starch solution dissolved in different buffer solutions for 20 min at 55 °C. The crude enzyme activity of each reaction was analyzed by DNSA assay. All experiments were conducted in triplicate and the results are expressed as mean ± SD.

2.12. Determination of the Effects of Different Metal Ions on the Extracellular Amylase Activity

For determining the effects of various metal ions on the extracellular amylase activity, CaCl₂, MgCl₂, MnCl₂, CuCl₂, FeCl₂, and HgCl₂ (Sigma Aldrich, Hamburg, Germany) were each individually mixed with crude extracellular amylase enzyme solution at a final concentration of 0.0015 M and incubated at 60 °C for 30 min. The extracellular amylase activity was then measured by DNSA assay as described previously in Section 2.9. All experiments were conducted in triplicate. The crude amylase activity in the absence of metal ions was used as the control and considered 100% activity.

2.13. Detection of Raw Cassava Starch Hydrolysis by Crude Extracellular Amylase of NW2 Isolate Using the Iodine–Starch Test

Cassava starch was purchased from a local market. Different concentrations of cassava starch (0.5%, 1%, 2%, 3%, and 4%,) suspension were prepared by dissolving it in 0.05 M sodium phosphate buffer (pH 8.0). An amount of 0.1 mL of crude enzyme was mixed with 0.9 mL of each different cassava starch suspension separately and incubated for 4 h at 60 °C. Then, 0.1 mL of iodine solution (2% I₂ and 0.2% KI) was added into each reaction mixture and mixed well to observe color differences. A control reaction prepared using 1% cassava raw starch solution without the addition of crude enzyme was included for comparison.

3. Results

3.1. Isolation and Identification of Thermophilic Bacterial Isolates That Produce Extracellular Amylase Enzyme

In this study, we isolated three thermophilic bacterial strains (NW2, NW3, and NW6) from the Nelumwewa geothermal spring in Sri Lanka. The thermal spring exhibited an average pH of 7.5, indicating slightly alkaline conditions, while average water temperature ranged from 48 °C to 55 °C across different sampling points. All isolates showed robust growth at 55 °C in NB as well as on nutrient agar plates at pH 7.5. Among the three bacterial isolates cultured on starch agar plates, only NW2 exhibited prominent clear zones around its colonies (Figure 2), with an average diameter of 25 mm, indicating a high potential for extracellular amylase production. In contrast, isolates NW3 and NW6 demonstrated only marginal amylase activity, with very narrow clear zones around their colonies (Figure 2). Therefore, due to the pronounced extracellular amylase activity of NW2 isolate, it was selected for further and detailed characterization of extracellular amylase enzyme activity. Moreover, NW2 isolate indicated extracellular protease activity as well (Figure 3). Light microscopy revealed that the NW2 isolate is a Gram-positive, rod-shaped bacterium (Figure 3). A comparison of morphological and biochemical characteristics of NW2 isolate with related bacterial species is summarized in the

Table 1. Comparison of morphological and biochemical characteristics of the NW2 isolate with those of already isolated Anoxybacillus species. (+ and − signs indicate positive and negative biochemical tests results, respectively. NR—Not reported).

Bacterial Characteristics	NW2	1 *	2 *	3 *	4 *	5 *	6 *	7 *
Optimal growth pH	7.5	7	7.0	8.0	5.6	7.0	7.0	7.0
Optimal growth temperature (°C)	60	70	60	50	61	55	60	60–65
Amylase activity	+	+	+	+	+	+	+	+
Protease activity	+	NR	−	−	−	NR	+	−
Catalase activity	+	−	NR	+	+	NR	+	+
Colony Color	Off-white	Golden brown	NR	White	Cream	NR	NR	Yellow
Gram Stain	+	+	NR	+	+	NR	NR	+
Cell shape	Rod	Rod	Rod	Rod	Rod	Rod	Rod	Rod
Culture medium	Nutrient broth	Luira–Bertani broth	Nutrient broth	Nutrient broth	Nutrient broth	Luira–Bertani broth	T5 medium	Anaerobic basal medium
Source of isolation	Thermal springs	Thermal springs	Thermal springs	Thermal springs	Thermal springs	Thermal springs	Thermal springs	Thermal springs

* 1—Anoxybacillus mongoliensis MBT001 [23]; 2—Anoxybacillus sp. AH1 [22]; 3—Anoxybacillus sp. KP1 [33]; 4—Anoxybacillus amylolyticus [34]; 5—Anoxybacillus thermarum A4 [35]; 6—Anoxybacillus sp. YIM 342 [36]; 7—Anoxybacillus flavithermus DSM 2641 [15].

.

3.2. Molecular Characterization of Thermophilic Bacterial Isolates

Based on 16S rRNA gene sequence analysis of the thermophilic bacterial isolates, isolate NW2 exhibited 99.3% identity to Thermaerobacillus caldiproteolyticus strain SF03 (NR_115200.1), whereas NW6 isolate showed 99.71% identity to Bacillus licheniformis strain ATCC 14,580 (NR_074923.1). Moreover, NW3 showed 96.85% identity to Anoxybacillus gonensis strain G2 (NR_025667.1) (

Table 2. Molecular characterization of thermophilic bacterial isolates by 16S rRNA gene sequencing.

Isolates	Accession Number 1	Closest Relative Bacterial Species 2	Accession Number 3	Identity 4
NW2	PV489057	Thermaerobacillus caldiproteolyticus strain SF03	NR_115200.1	99.30%
NW3	PV489066	Anoxybacillus gonensis strain G2	NR_025667.1	96.85%
NW6	PV489038	Bacillus licheniformis strain ATCC 14580	NR_074923.1	99.71%

¹ NCBI gene bank accession number of thermophilic bacteria isolated in this study. ² The closest bacterial species with the highest percentage identity match available in the NCBI gene bank. ³ NCBI gene bank accession number of the closest bacterial species. ⁴ BLAST search identity of the closest bacterial species available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 5 May 2025).

).

Phylogenetic analysis of the thermophilic bacterial isolates revealed that NW2, NW6, and NW3 are clustered into three separate groups A, B, and C, respectively (Figure 4). The NW2 isolate is grouped into cluster A, showing higher similarity to T. caldiproteolyticus species. Cluster B mainly consists of B. licheniformis strains, and the NW6 isolate is grouped into cluster B, exhibiting a close phylogenetic relationship with B. licheniformis. Moreover, the NW3 isolate is grouped into cluster C, with a higher similarity to A. gonensis.

3.3. Optimum Growth Temperature of the NW2 Isolate

The NW2 isolate exhibited optimal growth characteristics at 55 °C (Figure 5). The lag phase of the growth curve lasted for about 5 h and then reached the exponential phase. The bacterial growth rates at exponential phases were determined by linear regression analysis (R² > 0.95). The bacterial growth reached the stationary phase after about 11 h, indicating rapid growth characteristics at its optimum temperature. The NW2 isolate exhibited suboptimal growth at all other temperatures tested (Figure 5). Moreover, the maximum growth rate of the NW2 isolate was exhibited at 55 °C (Figure 6). These results indicate that the NW2 isolate is a moderate thermophile.

3.4. Characterization of Crude Extracellular Amylase Produced by NW2 Isolate

3.4.1. Determination of Optimal Temperature for the Enzyme Activity

To determine the optimal temperature of extracellular amylase produced by NW2 isolate, activity of crude amylase was analyzed by DNSA assay at different temperatures ranging from 40 °C to 80 °C. According to the results, the optimum temperature was determined to be 60 °C. However, the enzyme exhibited substantial activity up to 75 °C, which declined drastically thereafter (Figure 7). These results indicate that the extracellular amylase produced by NW2 isolates is thermostable in a wide range of temperatures from 55 °C to 75 °C.

3.4.2. Determination of Optimal pH Enzyme Activity

Moreover, the amylase enzyme exhibited optimal activity at pH 8.0 in both sodium phosphate and tris-HCl buffer systems (Figure 8). Furthermore, it remains substantially active within a pH range of 7.5 to 8.5. These findings suggest that the extracellular amylase functions effectively in slightly alkaline conditions.

3.5. Extracellular Crude Amylase Activity of NW2 Isolate Under Optimum Temperature and pH

The crude extracellular amylase was harvested from the overnight culture of NM2 isolate cultured in NB (60 °C and pH 8). By DNSA assay, the crude extracellular amylase activity was quantified revealing a mean activity of 0.85 U/mL under the optimal assay conditions (60 °C and pH 8).

3.6. Effects of Different Metal Ions on the Extracellular Amylase Activity of NW2 Isolate

The effects of various metal ions on the activity of crude extracellular amylase were analyzed and expressed as a percentage relative to the control activity. Crude amylase activity was significantly enhanced by Ca²⁺ and Mn²⁺ ions. Conversely, Mg²⁺, Cu²⁺, and Fe²⁺ ions slightly inhibited the enzyme activity. Moreover, the Hg²⁺ ion strongly inhibited the activity of crude amylase (

Table 3. Effects of metal ions on activity of the extracellular amylase of NW2 isolate.

Metal Ion (1.5 mM)	Relative Activity 1 (%)
Control (without any metal ions)	100
Ca2+	120 ± 5.2
Mn2+	116 ± 6.3
Mg2+	85 ± 4.5
Cu2+	80 ± 6.7
Fe2+	76 ± 5.5
Hg2+	5 ± 2.6

¹ Crude extracellular amylase activity was measured at 60 °C and pH 8 by DNSA assay as described in Section 2.9. Control activity (100%) was considered as 0.85 U/mL.

).

3.7. Hydrolysis of Raw Cassava Starch by NW2 Crude Extracellular Amylase

Under the optimum conditions, (pH 8.0 and temperature 60 °C), the crude amylase isolated from the NW2 isolate exhibited hydrolyzing of raw cassava starch (Figure 9), indicating its potential commercial application in the food, textile, and detergent industries.

4. Discussion

Extremophiles are increasingly recognized as valuable sources of industrially relevant enzymes due to their ability to produce extremozymes that remain active under extreme environmental conditions. Many thermally adapted enzymes or thermozymes, such as thermostable amylases, proteases, cellulases, and lipases, used in diverse industrial applications have been extensively derived from thermophilic and other extremophilic microorganisms [6]. Owing to their remarkable resilience and efficiency, extremozymes have enormous economic potential in the biotechnology sector. Among the microbial extremozymes, thermally stable amylases are of significant interest in all starch-related industries [6,37], while these enzymes account for nearly 25% of the world enzyme market [9]. As a result, thermozymes have received greater attention for their suitability in high-temperature industrial processes, where conventional enzymes often fail to perform optimally [19].

In the present study, three thermophilic bacterial strains were successfully isolated and molecularly characterized from the Nelumwewa geothermal spring in Sri Lanka. These isolates exhibited robust growth at 55 °C under slightly alkaline conditions (pH 7.5). However, among them, only NW2 isolate exhibited prominent extracellular amylase activity as well as extracellular protease activity, indicating it to be a promising candidate to produce thermostable industrial enzymes. Molecular characterization of NW2 isolate revealed that it shared 99.3% 16S rRNA gene sequence identity with Thermaerobacillus caldiproteolyticus strain SF03, previously classified as Anoxybacillus caldiproteolyticus or Geobacillus caldoproteolyticus [38,39], a well-documented thermophilic bacterium known for its thermostable enzyme production [40,41]. Phylogenetic analysis grouped the three isolates into three distinct clusters. The NW2 isolate clustered with T. caldiproteolyticus strains (Cluster A), indicating a close evolutionary relationship, while NW6 and NW3 were grouped into Clusters B and C, respectively, showing phylogenetic affiliation with Bacillus licheniformis and A. gonensis. A recent study on bacterial diversity analysis of Mahapelessa geothermal spring in Sri Lanka has identified some bacterial species such as Pannonibactor spp., Gulbenkiania spp., Pigmentiphaga spp., Enterobacter spp., Klebsiella spp., and Bacillus spp. [25]. However, it did not isolate any Thermaerobacillus spp., Anoxybacillus spp., or B. licheniformis_, underscoring the wide bacterial diversity across different geothermal springs in Sri Lanka.

Under optimal growth conditions (55 °C and pH 7.5), NW2 isolate reached the stationary phase after about 10 h in NB medium, indicating its rapid growth characteristics. Some reports have suggested that bacterial amylase production is growth dependent [42,43]. Moreover, NW2 extracellular amylase exhibited optimal activity at 60 °C and pH 8.0 which are higher than those of optimal bacterial growth. This is a common trait found in some thermostable enzymes due to their structural rigidity [44], suggesting evolutionary adaptation to the seasonal fluctuation of temperature and pH of geothermal springs. The optimal temperature and pH of the extracellular amylase activity of NW2 isolate are almost consistent with already reported amylase activity of some Anoxybacillus spp. [22,23]. Under these optimal conditions for the enzyme activity, the crude extracellular amylase unit activity of NW2 was found to be 0.85 U/mL. The unit activity of this enzyme is somewhat consistent with previous reports on crude amylase activity from thermophilic Anoxybacillus species [23].

Numerous studies have demonstrated that extracellular amylases isolated from various bacterial species possess significant potential for industrial-scale applications. Amylases derived from the thermophilic bacterium B. licheniformis exhibit excellent amylolytic properties and have been successfully applied in the biodegradation of food waste [45]. Moreover, amylases from different bacterial sources display remarkably high thermal optima, including 135 °C for B. subtilis [46] and 85 °C for Rhodothermus marinus [47]. Other thermophilic bacteria, such as Bacillus caldolyticus [48] and Geobacillus thermoleovorans [49], exhibit peak enzymatic activity at around 70 °C, highlighting the wide range of thermostability among bacterial amylases. The thermostability and broad pH tolerance of these enzymes reduce the need for cooling steps and pH adjustments, improving process efficiency and cost-effectiveness.

The bacterial species T. caldiproteolyticus (formerly A. caldiproteolyticus) is already known for its production of extracellular proteases rather than amylase [40]. To date, only a single study has reported the production of extracellular amylase by this bacterial species [50]. However, that study lacks comprehensive details regarding the activity of the extracellular amylase produced by A. caldiproteolyticus. Furthermore, the two known bacterial strains most closely related to the NW2 isolate have not provided detailed information regarding their amylase activities [40,50], suggesting the need for further studies to investigate the potential of producing extracellular amylase from this species.

The presence of different metal ions in the enzyme reaction mixture can significantly affect the activity and stability of thermostable amylases [51]. The crude amylase activity of the NW2 isolate was significantly enhanced by Ca²⁺ and Mn²⁺ ions. The Ca²⁺ ion has been reported to play a vital role in enhancing the activity of most of the thermostable amylases by maintaining the structural integrity and catalytic efficiency at higher temperatures [52]. However, it has been reported that the activity of some amylases is independent of Ca²⁺ [14]. Moreover, the Hg²⁺ ion strongly inhibited the activity of crude amylase of NW2. This may be due to binding to the active site of the enzyme or altering the correct three-dimensional conformation of the enzyme.

Cassava starch is extensively utilized across diverse industries, including food processing, textiles, and bioethanol production. However, its hydrolysis is often associated with high operational costs, primarily due to the energy-intensive nature of conventional processing methods. Recent studies have demonstrated the successful application of microbial amylases in the production of bioethanol from raw cassava starch [53]. In this context, the extracellular amylase produced by the NW2 isolate in the present study exhibited strong hydrolytic activity toward raw cassava starch, highlighting its potential for use in both the food and biofuel industries, where efficient and rapid starch degradation is essential. Furthermore, the enzyme’s stability under moderately alkaline conditions enhances its applicability in the detergent industry, where alkaline-tolerant amylases are required.

To the best of our knowledge, this is the first report of a T. caldiproteolyticus-like strain from Sri Lankan geothermal springs exhibiting a remarkable extracellular amylase activity. The NW2 isolate is a Gram-positive, rod-shaped, and catalase-positive bacterium. These morphological and biochemical characteristics are commonly associated with well-known amylase-producing bacteria [54]. In contrast, the NW6 isolate exhibited a very close phylogenetic relationship with 99.71% 16S rRNA gene sequence identity with B. licheniformis, a well-established industrial bacterial species recognized for its thermostable amylase production [55,56,57,58]. However, NW6 did not exhibit significant extracellular amylase activity under the tested conditions, suggesting potential strain-level variability or previously undocumented functional diversity within the species.

Overall, the findings of the present study not only reveal the biotechnological potential of the bacteria isolates from the Nelumwewa geothermal spring but also highlight the geothermal springs of Sri Lanka as a promising niche environment for exploring thermophilic enzyme-producing microbes.

5. Conclusions

This study highlights the biotechnological potential of NW2, a T. caldiproteolyticus-like strain from the Nelumwewa geothermal spring in Sri Lanka, a previously unexplored thermal ecosystem. The NW2 isolate emerged as a potential candidate for industrial applications because of its prominent extracellular amylase production with notable thermostability and activity in slightly alkaline environments. Furthermore, the extracellular amylase of NW2 demonstrated effective hydrolysis of raw cassava starch, suggesting its direct applicability in food, bioethanol, and detergent industries where robust and efficient enzymatic performance under high temperature and pH is critical. This marks the first report of a T. caldiproteolyticus-like strain from Sri Lanka with such enzymatic activity, expanding the known functional diversity of this species and underscoring the potential for strain-level variation. Moreover, this is the first detailed report characterizing the extracellular amylase from T. caldiproteolyticus. The findings of the present study reinforce the value of geothermal environments as reservoirs of novel extremozymes, suggesting further exploration to support the development of sustainable, high-performance biocatalysts for industrial applications.