Characterization and biotechnological application of protease from thermophilic Thermomonas haemolytica

Abstract

This study aimed to determine the protease enzyme production capability of Thermomonas haemolytica, an isolate from the geothermal Nenehatun hot spring in Turkey, and to explore the potential use of this enzyme in the detergent industry for removing protein-based stains. Protease-producing strains were screened from hot spring samples, and a promising strain was identified as T. haemolytica based on its morphological, physiological, and biochemical characteristics, as well as the sequence of its 16S rRNA gene. Maximum protease activity was observed at a temperature of 55 °C and a pH of 9.0 after 72 hours of incubation. The enzyme demonstrated significant stability between 50 and 65 °C and across a pH range of 8.0 to 10.0. The enzyme activity was strongly inhibited by PMSF and partially inhibited by EDTA, EGTA, SDS, and urea. Certain divalent metal ions, including Ca2+, Mg2+, and Mn2+, enhanced the enzyme activity, while Zn2+ and Cu2+ caused a decrease. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) values were determined using the Lineweaver-Burk plot as 125 EU/ml and 1262 mg/ml, respectively. The biochemical characteristics of the protease obtained from T. haemolytica were analyzed, and the enzyme was applied to fabrics stained with blood and grass in conjunction with detergent to assess its stain removal effectiveness. The results indicated that the use of detergent with the enzyme was more effective in cleaning the stained fabrics compared to using detergent alone. This study represents the first characterization of the protease from T. haemolytica. The findings suggest that this novel strain holds promise for industrial applications in the production of detergents.

Introduction

Thermophilic microorganisms are highly favored in biotechnological enzyme production due to the ability of their enzymes to catalyze biochemical reactions at elevated temperatures. Consequently, numerous studies have focused on thermophilic enzymes. These enzymes exhibit a significantly longer shelf life compared to mesophilic enzymes and possess greater stability across a wider range of temperatures and pH values, making them less susceptible to decomposition and denaturation by organic solvents.

Protease enzymes function by hydrolyzing proteins into free amino acids and peptides, leading to their widespread application across diverse industrial sectors. Proteases are utilized in the detergent, food, leather, meat, and milk industries, as well as in photographic applications, pet food production, medical applications, and molecular research. Notably, the detergent industry represents one of the most promising areas for the extensive use of protease enzymes. Given their resilience at high temperatures and pH levels, thermophilic proteases are particularly valuable as stain removal agents. Therefore, the incorporation of proteases can significantly contribute to more environmentally friendly practices within the detergent industry by reducing the necessity for toxic substances.

Over the past two decades, considerable research has been dedicated to the isolation, purification, and characterization of protease-producing bacterial strains. However, there has been no prior investigation into thermophilic bacteria and their enzymes derived from the Nenehatun hot spring located in Erzurum. This study aimed to identify the bacterial isolate exhibiting the highest protease activity and to evaluate the biochemical properties and potential application of the bacterial enzyme in the detergent industry.

Materials and methods

Isolation of bacteria

Water and sludge samples were collected from the Nenehatun hot spring in Erzurum, Turkey. These samples were serially diluted in concentrations ranging from 10−1 to 10−5 and spread onto Nutrient Agar (NA) plates. The plates were then incubated under both aerobic and anaerobic conditions at temperatures between 45 and 60 °C for a period of 24 to 48 hours. Following incubation, colonies displaying distinct characteristics such as shape and color were selected and purified through subculturing. The purified bacterial strains were preserved in Luria-Bertani Broth containing 15% glycerol at −80 °C for subsequent experimentation.

Screening of protease activity of the bacterial isolates

The purified bacterial isolates were streaked onto an agar medium supplemented with 10% skim milk and incubated at 55 °C for 48 to 72 hours. Isolates capable of producing protease were identified by the formation of a transparent zone around their colonies on the medium. The isolate exhibiting the largest zone of proteolysis was selected for further investigation.

Identification of morphological, physiological, and biochemical characteristics of bacterial isolate with protease activity

The morphological characteristics of the cells and colonies, Gram staining, spore staining, and the presence of catalase, oxidase, and amylase activities were determined using standard microbiological methods. The selected isolate was subjected to incubation at various temperatures (20 to 70 °C), pH levels (3 to 11), and sodium chloride (NaCl) concentrations (2 to 12%) to determine the optimal conditions for bacterial growth. The optimal incubation time was assessed at intervals between 12 and 76 hours. The optimum values for incubation time, temperature, pH, and NaCl concentration for bacterial growth were determined by measuring the absorbance of bacterial cultures at 600 nm. The isolate’s requirement for atmospheric oxygen was assessed by incubation in Brain Heart Infusion Agar media. The carbon sources utilized by the isolate were also identified.

Analyse of 16S rRNA gene region

DNA isolation was performed using a modified method. The isolated DNA was stored at −80 °C for further analysis. The 16S rRNA gene region of the isolate demonstrating protease activity was amplified using universal primers. The PCR reaction was conducted under specific conditions. The PCR product was cloned into a pGEM®-T Easy vector system, and the recombinant vector was introduced into Escherichia coli JM101 using the CaCl2 method. Plasmid isolation was subsequently performed using a commercial plasmid DNA extraction kit. The 16S rRNA gene was then sequenced by a commercial sequencing service, and the resulting sequence was compared with sequences in GenBank using the BLAST search tool.

Determination of protease enzyme activity

To produce the protease enzyme, the bacterial isolate was inoculated into a medium containing 5 g/L casein, 5 g/L peptone, 2 g/L yeast extract, 5 g/L NaCl, 0.2 g/L MgSO4·7H2O, 0.1 g/L CaCl2, and 1 g/L K2HPO4. The pH of the medium was adjusted to 9 using a 10% Na2CO3 solution. The bacterial culture was incubated at 55 °C with shaking at 150 rpm for a duration of three days. The protease activity was measured on the first, second, and third days to determine the optimal incubation time for enzyme production. Following incubation, the culture was centrifuged at +4 °C and 5000 rpm for 20 minutes. The resulting supernatant, free of cell debris, was used as the enzyme solution for subsequent enzyme activity experiments. For these experiments, 0.5 ml of the enzyme solution was mixed with 2.5 ml of a 0.6% casein solution prepared in a 50 mM glycine-NaOH buffer at pH 9. The mixture was incubated at 55 °C for 20 minutes to allow the protease enzyme to act. The enzyme reaction was then stopped by the addition of 2.5 ml of a 0.1 M trichloroacetic acid (TCA) solution, and the mixture was further incubated at 37 °C for 30 minutes. After this incubation, the reaction solution was filtered, and 2.5 ml of a 0.5 M Na2CO3 solution and 0.5 ml of 1 N Folin-Ciocalteu’s phenol reagent were added to the filtrate. Following a 30-minute incubation at room temperature, the absorbance of the resulting solutions was measured at 660 nm. One unit (EU/ml) of protease activity was defined as the amount of enzyme required to produce 1 μg of tyrosine per minute through the breakdown of the casein substrate.

The effects of temperature on enzyme activity and the thermal stability of the enzyme

To determine the influence of temperature on enzyme activity, the enzyme activity of the culture supernatant was measured across a temperature range of 35 to 75 °C. The highest observed activity was set as 100%, and the activities at other temperatures were expressed as relative percentages. To assess the thermal stability of the enzyme, it was incubated in glycine-NaOH buffer at 40 °C, 50 °C, 55 °C, and 60 °C for periods of one, two, and three days.

The effects of pH on enzyme activity and pH stability

To investigate the impact of pH on enzyme activity, casein substrate solutions with pH values ranging from 6 to 13 were used. To evaluate the pH stability of the enzyme, it was incubated in buffer solutions with pH values of 8.0, 9.0, 10.0, and 11.0 at 30 °C for durations of one, two, and three days.

The effects of metal ions and inhibitors on the activity

To examine the influence of various divalent metal ions, including MnCl2, ZnSO4·7H2O, MgCl2, FeCl3, CuSO4, and CaSO4, on enzyme activity, the culture supernatants were incubated in buffer solutions containing 5 mM of each of these ions for a period of two hours. The relative activities compared to a control sample were then calculated. To determine the effects of 5 mM EDTA, EGTA, DTT, PMSF, urea, and 1% SDS, Tween 20, Tween 80, Triton X-100, and H2O2 on enzyme activity, the same volume of culture supernatant was mixed with these solutions and incubated at 37 °C for two hours.

Determination of Km and Vmax Values

To determine the effect of substrate concentration on enzyme activity, casein solutions with concentrations of 0.5, 1, 2, 4, 6, and 8 mg/ml were prepared. The results were analyzed by plotting the reciprocal of the reaction velocity (1/V, in EU/ml) against the reciprocal of the substrate concentration (1/S, in mg/ml of casein). The Michaelis-Menten constant (Km) and the maximum velocity (Vmax) values were then determined from these Lineweaver-Burk plots.

Biotechnological applications with protease enzyme

Sterilized cotton cloths stained with blood and grass were individually immersed in sterilized water, culture medium, detergent solution, culture supernatant, and a mixture of detergent and culture supernatant to assess the stain removal capability of the culture supernatants. Visual comparisons were made before and after the application to evaluate the effectiveness.

Results

Isolation and determination of morphological, physiological, and biochemical characteristics of bacteria

A total of 85 bacterial isolates capable of growth at 45 °C and 60 °C were obtained. The isolate exhibiting the highest protease activity was selected for further study.

16S rRNA gene sequence analysis

Sequence analysis of the 16S rRNA gene of the selected isolate revealed a sequence of approximately 1489 nucleotides. Following successful sequencing and alignment using BLAST, the bacterium was identified as Thermomonas haemolytica with a similarity rate of 97%. This sequence information has been deposited in Genbank under the accession number MG016525.

Protease enzyme activity

Investigation of protease activities among the isolates identified the highest activity in Thermomonas haemolytica IY13, measuring 101.33 EU/ml. The optimal temperature range for the enzyme activity of T. haemolytica IY13 was found to be between 50 and 65 °C, with an optimum temperature of 55 °C. Incubation of T. haemolytica IY13 isolate for one day resulted in decreases in temperature stability of 5%, 18%, 20%, and 30% at 40 °C, 50 °C, 55 °C, and 60 °C, respectively. On the second day of incubation, these decreases were measured as 28%, 32%, 45%, and 80% at the same respective temperatures. By the end of the third day, significant drops in activity were recorded as 75%, 70%, 95%, and 100% at 40 °C, 50 °C, 55 °C, and 60 °C. The optimal pH range for the T. haemolytica IY13 isolate was determined to be between 8 and 12. The enzyme maintained 100% of its stability at pH 8, 9, and 10, and 90% stability at pH 11 after one day. On the second day, the activities were 92%, 85%, 85%, and 77% at pH 8, 9, 10, and 11, respectively. By the third day, the activities were further reduced to 72%, 72%, 70%, and 60% at the same pH values. Incubation of T. haemolytica IY13 with EDTA, EGTA, DTT, PMSF, urea, SDS, Tween 20, Tween 80, Triton X-100, and H2O2 separately resulted in relative enzyme activities of 80%, 97%, 100%, 25%, 90%, 78%, 119%, 125%, 92%, and 98%, respectively. The effects of metal ions revealed that Ca2+, Mg2+, and Mn2+ ions increased enzyme activity by 120%, 118%, and 130%, respectively, while Zn2+, Cu2+, and Fe3+ ions had an opposite effect, reducing activity to 95%, 80%, and 91%, respectively. Based on Lineweaver-Burk plots, the Km value for T. haemolytica IY13 was calculated as 1262 mg/ml, and the Vmax value was determined as 125 EU/ml.

Discussion

This study represents the first investigation of the Nenehatun hot spring and the identification of a new bacterial species, Thermomonas haemolytica, in Turkey. Furthermore, the protease enzyme produced by this bacterium has been investigated for the first time for its potential biotechnological applications. The optimal temperature and pH for the T. haemolytica IY13 isolate were determined to be 55 °C and pH 9.0, respectively, classifying the obtained protease as a thermophilic alkaline protease. This characterization is consistent with findings reported in existing literature for similar enzymes. The T. haemolytica IY13 isolate lost 70% of its enzyme activity after a three-day incubation at 50 °C and 28% of its activity at pH 9.0. Despite the relatively long three-day incubation period, the protease enzyme from this isolate exhibited considerably high stability. The pH stability of protease enzymes is particularly important for their industrial application in detergents. The observed stability of the enzyme activity at pH 8.0 and 9.0, with activity remaining above 50% even after prolonged incubation, is a significant finding. Considering these factors, it is anticipated that protease enzyme products derived from this isolate would possess a favorable shelf life.

Numerous studies on protease enzyme activity have generally reported negative impacts from PMSF, EDTA, EGTA, SDS, DTT, H2O2, and urea. Conversely, Tween 20, Tween 80, and Triton X-100 have often been found to have minimal or even positive effects on enzyme activity. Our findings revealed that PMSF significantly inhibited the protease activity of T. haemolytica IY13, suggesting that the identified enzyme may belong to the serine protease group, as PMSF is known to inhibit serine residues in the active sites of these enzymes. The observed loss of enzyme activity in the presence of EDTA indicates the potential involvement of metal ions in the enzyme’s structure or function, suggesting it might also possess characteristics of a metalloprotease. The high stability of the alkaline protease against H2O2, an oxidizing agent, is a desirable trait for its incorporation into whitening detergent formulations. The minimal 2% decrease in enzyme activity of the protease from T. haemolytica IY13 in the presence of H2O2 suggests its potential suitability for the detergent industry.

Alkaline proteases are known to require metal ions for maintaining their stability at high temperatures and preserving their active conformations. Studies have also indicated that Ca2+, Mg2+, and Mn2+ ions typically enhance enzyme activities, while Cu2+ and Zn2+ ions often reduce them. In this study, the effects of Ca2+, Mg2+, Mn2+, Zn2+, Cu2+, and Fe3+ ions on the protease enzyme were investigated. The results showed that Ca2+, Mg2+, and Mn2+ ions increased enzyme activity, supporting their role as potential cofactors, whereas Zn2+ and Cu2+ ions decreased activity.

Although the protease enzyme obtained from the T. haemolytica IY13 isolate was not purified, its biotechnological application was explored to assess the potential use of the crude enzyme solution in the detergent industry. The application of the casein-induced enzyme solution to blood and grass-stained cotton cloths, both alone and in combination with detergent, demonstrated that the mixture of crude enzyme solution and detergent was more effective in removing stains compared to detergent or water alone. Consistent with existing literature, several bacterial-based protease enzymes have shown efficiency in removing stains from cotton fabrics. Therefore, it is proposed that the protease from this isolate could be purified and developed into an effective formulation for stain removal in detergent industries.