Optimization, partial purification, and characterization of a novel high molecular weight alkaline protease produced by Halobacillus sp. HAL1 using fish wastes as a substrate
Journal of Genetic Engineering and Biotechnology volume 21, Article number: 48 (2023)
Hydrolytic enzymes from halophilic microorganisms have a wide range of industrial applications. Herein, we report the isolation of Halobacillus sp. HAL1, a moderately halophilic bacterium that produces a novel high molecular weight extracellular alkaline protease when grown in fish processing wastes as a substrate.
Results showed that the isolated strain belonged to the genus Halobacillus, and it was designated as Halobacillus sp. HAL1 with the GenBank accession number OK001470. The strain secreted an extracellular alkaline protease, and the highest yield was obtained when it was grown in a medium with fish wastes substrate as the sole nutritional source (10 g/L) and incubated at 25 °C under shaking conditions. The enzyme was partially purified by Sephadex G-100 column chromatography. Zymographic analysis showed two casein degrading bands of about 190 and 250 KDa. The optimum enzyme activity was at a temperature of 50 °C at pH 8. The proteolytic activity was enhanced in the presence of metal ions (Ca2+, Mg2+, and Mn2+), surfactants (Tween 80, SDS, and Triton-X100), H2O2, and EDTA.
Our study indicates that Haobacillus sp. HAL1 is a moderately halophilic strain and secrets a novel high molecular wight alkaline protease that is suitable for detergent formulation.
Microorganisms are a valuable source of enzymes for both industrial and medical uses because of their rapid growth rate and simplicity of manipulation, especially with the advent of recombinant DNA technology and protein engineering . Microbial enzymes have been employed in the catalytic bioprocesses of a variety of industries, including food, agriculture, chemicals, medicine, and energy. Microbial enzymes are preferred over plant and animal enzymes in industry and medicine due to their stability, higher catalytic activity, regular supply, greater yield, and lower cost of recovery from the producing microbes. Furthermore, as compared to traditional catalytic methods, microbial enzymes perform well under a wide range of chemical and physical conditions, are more efficient, produce high-quality products, and are less harmful to the environment [2, 3]. The development of novel, economically competitive, and sustainable production processes necessitates the rapid discovery of novel enzymes with unique properties .
Due to their highly flexible metabolism, extremophilic bacteria have adapted to survive in extreme conditions (e.g., high/low temperature, pH, salinity, and pressure) and are potential sources of catalytically stable enzymes (proteases, amylases, lipases…etc.) that could work under harsh industrial conditions [5,6,7] and therefore, are attractive for different industries, especially those including high salt concentrations, such as textile, fermented food, pharmaceuticals, cosmetics, and leather industries [8,9,10].
Proteases are widely used in the food, detergent, and pharmaceutical industries. They represent about 60% of the industrial enzyme market, with increasing global demand during 2014–2019 at a compound annual growth rate (CAGR) of 5.3% and is expected to increase significantly as they become more widely applied in bioremediation and the leather processing industries [3, 11]. Microbial proteases account for about two-thirds of commercial proteases because they have the characteristics required for industrial applications (e.g., less time consumption, high yield, cost-effectiveness, less space requirement, and genetic manipulation) compared to plant and animal proteases [12, 13]. Microbial proteases are classified into acidic and alkaline proteases based on their pH range of activity. Acidic proteases are active at acidic pH, while alkaline proteases are active at alkaline pH. Among microorganisms, bacteria are the primary source of alkaline proteases, with the Bacillus genus being the most prolific producer and the most commercially exploited microbes .
The high cost of substrates is the most important factor limiting the production of microbial enzymes for industrial applications; thus, using low-cost substrates is important from a commercial standpoint [14, 15]. The search for low-cost substrates suitable for microbial enzyme production is critical [15,16,17]. Fish processing waste (FPW) is a low-cost nutritional substrate that is suitable for the growth of enzyme-producing microorganisms and can be used to produce enzymes. Several studies reported the use of FPW to produce microbial enzymes .
Sabkhas or “salt falts” are saline environments that are periodically inundated with water, and evaporites are formed due to capillary evaporation . Microorganisms that are halophilic or halotolerant inhabit these environments . Despite the prevalence of sabkha environments along the Egyptian Red Sea coast, little is known about the microorganisms inhabiting these environments. The sabkha of wadi abu-Shaar, located north of Hurghada City on the Egyptian Red Sea coast, is one of these environments that has not been extensively studied. The current study focuses on the optimization of production, partial purification, and characterization of a high molecular weight alkaline protease produced by Halobacillus sp. HAL1, which was recently isolated from the saline soil of wadi abu-Shaar, north of Hurghada City on Egypt’s Red Sea coast.
Wadi Abu-Shaar is about 10 km north of Hurghada City, between the latitudes of 27°18′25′′N and 33°43′15′′E (Fig. 1a). In the backshore area, there are sabkha evaporites and dwarf sand dunes covered in rare plants (Fig. 1b). Five different sediment samples were collected using a sterile scooper from the top 10 cm of saline soil and stored at 4 °C in sterile polyethylene bags. Within a few hours, the samples were transported to the laboratory and processed.
Isolation of heterotrophic bacteria
For isolation of salt-tolerant heterotrophic bacteria, 1 g of each sediment sample was homogenized in 9 mL of sterilized seawater and serially diluted up to 10−5. The serial dilutions of all samples were plated in tryptone soya agar medium prepared using artificial seawater containing 80 gm/L of NaCl. The plates were incubated for 24 h at 37 °C. One plate yielding well isolated colonies from each sample was selected, and one colony from each morphotype was picked and purified by streaking 2–3 times to ensure purity of the isolates. Pure cultures were stored at 4 °C . Six bacterial isolates (HAL1-HAL6) were obtained and screened for alkaline protease production.
Screening for alkaline protease production
To evaluate the proteolytic activity of the bacterial isolates, Horikoshi-I alkaline medium  was used with some modifications. The alkaline agar medium (pH 9) contained (g/L): glucose 10, peptone 5, yeast extract 5, Mg2SO4. 7H2O 0.2, K2HPO4 1, NaCl 50, Na2CO3 10, and agar 15. Skim milk (10%, v/v) was supplemented to the medium as an indicator for proteolytic activity. Glucose and Na2CO3 solutions were autoclaved separately, cooled down, and added to the autoclaved medium. Sterile filter paper discs, 5 mm in diameter, were impregnated separately with 30 μL of lag phase cultures of each isolate and put on the alkaline agar medium plates. The plates were incubated for 24 h at 35 °C, and the appearance of clear zone around the colonies was taken as evidence for the production of alkaline protease .
Identification of bacteria
The bacterial isolate (HAL1) with the highest alkaline protease production was identified based on its morphological and biochemical characteristics as described in Bergey’s manual of determinative bacteriology . The isolate was further identified using 16 s rDNA sequence analysis. Genomic DNA was extracted using the Hipura Bacterial DNA Kit (Angen Biotech, China) according to the manufacturer’s instructions. PCR amplification of the 16 s rDNA was carried out using the forward primer: 16F 27 (5′-AGA GTT TGA TCC TGG CTC AG-3′), and the reverse primer: 16R 1525 (5′-AAG GAG GTG ATC CAG CCG CA-3′). The PCR product was purified using the QIA quick gel extraction kit (Qiagen, USA) and sequenced using an automated sequencer (Macrogen, Korea). The identity of the isolate was determined by aligning the obtained sequence with the reference sequences available on the NCBI homepage using the BLAST algorithm (www.ncbi.nlm.nih.gov/blst). Multiple alignments and phylogenetic tree construction were performed using the Neighbor-Joining method using Mega-X software, version 10.1.7 .
To study the optimal growth conditions, the HAL1 strain was grown in the medium described above, without the addition of agar. The amount of NaCl was investigated in the range of 0–25%. The pH range was 4–10, and the temperature was 10–45 °C. All experiments were carried out in triplicate with shaking at 150 rpm. The bacterial growth was monitored by measuring the absorbance at 600 nm .
Preparation of fish wastes substrate
Fish by-products (viscera and head contents) of Scarus collana were obtained from a fish market in Hurghada, Egypt. To obtain fish wastes flour, viscera and head contents were cooked until boiling. The cooked materials were pressed to remove water and oil, dried for 24 h at 80 °C, and grinded .
Production of alkaline protease
The effectiveness of fish waste flour as a substrate for the production of HAL1 protease was tested using the following media:
YT medium (g/L): Peptone 10, yeast extract 1, K2HPO4 1, MgSO4. 7 H2O 1, MnSO4. 7 H2O 0.1, glucose 2 ; SCG medium (g/L): Scarus collana (SC) waste flour 10, K2HPO4 1, MgSO4. 7 H2O 1, MnSO4. 7 H2O 0.1, glucose 2; SC medium (g/L): SC flour 10. Synthetic seawater containing 90 g/L of NaCl was used to prepare all of the culture media. A volume of 50 mL of each medium was inoculated with 0.1 mL of HAL1 isolate suspension (A600 nm = 0.4) in 250 mL Erlenmeyer flasks, pH 9, and incubated for 24 h at 35 °C with shaking at 150 rpm. To maintain the alkaline condition, each culture was buffered with 50 mM Tris–HCl buffer (pH 9).
Determination of enzyme activity
Protease activity was measured according to previously described methods by Cupp-Enyard  with some modifications. Briefly, the reaction system (1.0 mL) composed of 250 μL of 0.65% casein in 100 mM Tris–HCl buffer (pH 8) and 250 μL of appropriately diluted cultivated supernatant, which was incubated for 30 min at 37 °C. The reaction was terminated by adding 500 μL of trichloroacetic acid (110 mM). Then it was centrifuged for 10 min at 10,000 rpm. Five hundred microliters of Na2CO3 (500 mM) and 0.3 of appropriately diluted Folin-Ciocalteu reagent were added to 0.2 mL of the supernatant, mixed thoroughly, and incubated for 30 min at 37 °C. To determine the amount of tyrosine liberated from the substrate, the absorbance of all sample replicates and the blank (containing deionized water instead of the enzyme solution). was measured at 660 nm using a JEN-WAY 6800 spectrophotometer. A standard curve was developed using tyrosine (0–16 μg/mL) as a standard substance. The absorbance response (y) of tyrosine (y = 0.0708x + 0.0056, R2 = 0.9894) and concentrations (0–16 μg/mL) was linear. One unit of enzyme activity was defined as the quantity of protease that liberates 1 μg/mL of tyrosine per min.
Optimization of alkaline protease production
Effect of NaCl concentration
To determine the optimum concentration of NaCl for alkaline protease production by strain HAL1, the strain was grown in SC medium supplemented with different concentrations of NaCl (1–15%) and incubated for 24 h at 30 °C under shaking conditions (150 rpm). All of the experiments were carried out in triplicate and the protease activities were determined.
Effect of fish waste substrate concentration
The effect of the concentration of Scarus collana waste flour on alkaline protease production was investigated at a range of 5–40 g/L, keeping all other parameters constant.
Effect of pH, temperature, and aeration
The influence of initial pH on alkaline protease production by strain HAL1 was investigated by culturing the strain in SC medium with different pH (6-11) while keeping all other parameters constant. Similarly, to investigate the effect of temperature on the alkaline protease bioprocess, the strain was grown under various temperatures (20–40 °C) at the optimum growth parameters. The effect of aeration on protease production was also investigated by growing the strain under static and shaking (150 rpm) conditions . All of the experiments were done in triplicate, and the enzyme activities were assayed as described above.
Fermentation and partial purification of the enzyme
Strain HAL1 was grown at pH 8 in a protease production medium prepared using artificial sea water and containing SC waste powder (10 g/L) and NaCl (3 g/L), with pH 8. The medium was autoclaved for 20 min at 121 °C, cooled to room temperature, and inoculated with 0.5% (v/v) of a 24-h-old culture of HAL1. The Fermentation process was carried out under shaking conditions (150 rpm) for 36 h at 25 °C. To separate the biomass, the culture was centrifuged at 4 °C for 10 min at 10,000 rpm. The enzyme was precipitated from the supernatant by adding two volumes of chilled acetone. The precipitated protein was dissolved in phosphate buffer (pH 8).
Gel filtration chromatography
Concentrated enzyme solution was applied onto gel filtration Sephadex G-100 column (ID 0.8 cm × BH 84 cm) previously equilibrated with 0.1 M Tris–HCl buffer pH 8. The column was eluted with 0.1 M Tris–HCl buffer pH 8. The flow rate was maintained at 20 mL/h and fractions of 3 mL were collected and dialyzed against the same buffer. The content of protein and the activity of the enzyme were determined for each fraction, and the fraction with the highest activity per mg of protein was chosen for enzyme characterization and zymography development.
Zymogram analysis was performed according to Garcia-Carreno et al.  with some modifications. Briefly, samples were separated on a 10% resolving gel supplemented with 0.1% casein as a copolymerized substrate. After separation, the gel was rinsed with distillated water and agitated in phosphate buffer (pH 8.3) containing Triton X-100 (2.5%) for 60 min. Subsequently, the gel was incubated in phosphate buffer (pH 8.3) at 50 °C for 12 h. Finally, the gel was stained with Coomassie brilliant blue R-250. The presence of alkaline protease activity was indicated by the appearance of a clear zone on a dark blue background.
Characterization of the enzyme
Effect of pH, temperature, and salt concentration
Various buffers (50 mM) with different pH values (3-11) were used to test the effect of pH on protease activity: citrate buffer (pH 3–6), phosphate buffer (pH 6–8), Tris–HCl (pH 8–9), and glycine–NaOH (pH 9–11). Fifty microliters of the enzyme was added to 200 µL of the appropriate buffer and mixed with 250 µL of 0.65% casein dissolved in distillated water, and the assay was carried out as described above. To determine the optimum temperature for protease activity, the assay was carried out at various temperatures (5–80 °C). The assay was performed in the presence of 0–4 M NaCl or 0–3 M KCl at the optimal pH and temperature to determine the effect of salt concentration on protease activity .
Effect of organic solvents
The effect of various organic solvents, including methanol, ethanol, xylene, acetone, hexane, benzene, propanol, and butanol, on protease activity was investigated. The partially purified enzyme was pre-incubated with each organic solvent at a 25% final concentration in 50 mM tris–HCl buffer (pH 8) for 10 min at 37 °C, and the assay was performed as described above. The activity of the enzyme was assumed to be 100% in the absence of organic solvent .
Effect of surfactants, EDTA, and H2O2
The effect of surfactants (SDS, Tween 80, and Triton X-100) and H2O2, on protease activity was determined by pre-incubating the enzyme with different concentrations (1, 5, and 10%) of the respective agent for 10 min. The effect of different concentrations of EDTA (1, 5, and 10 mM) on protease activity was studied by pre-incubating the enzyme with EDTA for 10 min. Protease activity was carried out for 1 h at the optimum pH and temperature and assayed as mentioned above. The enzyme activity without any surfactant, H2O2, or EDTA was regarded as 100% .
All experiments and assays were carried out in triplicates. Means and standard deviations were calculated by Microsoft Excel software.
Isolation of bacteria and screening for alkaline protease production
In the present study, the isolation of salt-tolerant heterotrophic bacteria from saline sediment yielded six bacterial isolates (HAL1-HAL6). Using alkaline agar medium supplemented with skimmed milk and 9% NaCl, all of the isolates were screened for the production of alkaline protease. Due to the highest proteolytic activity of the HAL1 isolate in comparison with the other isolates, it was chosen for further characterization and alkaline protease production (Fig. 2).
Morphological and biochemical characterization
Morphological and biochemical characterization of the HAL1 isolate revealed that colonies were yellow orange, smooth, circular, slightly raised, and approx. 2 mm in diameter after incubation for 2 days at 30 °C on nutrient agar supplemented with 10% NaCl (w/v). The pigment was not soluble in water and non-diffusible. HAL1 grew in a wide range of NaCl concentrations (1–21%, w/v) and optimally at 9% (w/v) NaCl. Furthermore, the isolate was aerobic, spore-forming, and grew optimally at 35 °C. The morphological and biochemical characteristics of the HAL1 isolate and the closely related bacterial species are depicted in Table 1.
The 16 s rRNA gene was amplified using PCR to determine the taxonomic position of the strain HAL1. The 1226 pb amplified product (Fig. 3) was sequenced and compared to the sequences in the NCBI nucleotide database using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/). The BLAST search revealed that the strain belongs to the genus Halobacillus and exhibited high similarity to many species of the genus: H. trueperi strain DSM 10404 (GenBank accession no. NR_025459, 99.87% similarity), H. karajiensis strain DSM 14948 (GenBank accession no. AJ486874, 99.74% similarity), H. dabanensis strain D-8 (GenBank accession no. NR_042860, 99.74% similarity), and H. faecis strain NBRC 103569 (GenBank accession no. NR_114247, 99.61% similarity). The sequence was deposited in the GenBank as Halobacillus sp. strain HAL1 with an accession number of OK001470. Figure 4 shows the phylogenetic relationship with the related Halobacillus spp. Paenibacillus polymyxa strain DSM 36 T was used as an outgroup to root the tree.
Production of alkaline protease
To test the suitability of S. collana waste substrate for alkaline protease production by strain HAL1, three culture media were used, all of which were made with synthetic seawater: YT medium, which contained commercial substrates, and two other media (SCG and SC), which were made with fish waste substrate flour. The results revealed that the SC medium, which contained only fish waste flour, supported higher levels of protease production (20.2 ± 0.40 U/mL) than the other media (Fig. 5). Protease activity was 17.71 ± 0.41 U/mL in the SCG medium, which contained both fish waste flour and glucose, and 14.87 ± 0.41 U/mL in the YT medium, which contained yeast extract and peptone. Based on these findings, SC medium prepared using synthetic seawater and containing only S. collana waste flour was chosen for optimization of the alkaline protease bioprocess by the HAL1 strain.
Optimization of protease production
Effect of NaCl concentration
The effect of sodium chloride concentration (1–15%) on the production of alkaline protease by strain HAL1 was studied. The strain produced the highest amount of protease in the medium containing 3% NaCl. However, a further increase in NaCl concentration caused a drastic decrease in alkaline protease production (Fig. 6).
Effect of substrate concentration
The effect of varying concentrations of S. collana waste flour on the production of alkaline protease was investigated at a range of 5–40 g/L. The results showed that at a concentration of 10 g/L, high alkaline protease production was achieved. However, a further increase in the concentration of fish waste flour caused a decrease in protease production (Fig. 7).
Effect of pH, temperature, and aeration
Figure 8 shows the effect of the incubation temperature (25–40 °C) on HAL1 protease production. The optimum temperature for the production of alkaline protease was found to be 25 °C (13.63 U/mL). With increasing growth temperature, there was a slight decrease in enzyme production, with the enzyme yield dropping to 10.41 U/mL at 40 °C. With regard to the pH effect, strain HAL1 was able to produce alkaline protease over a wide pH range (6-10) with maximum enzyme production at pH 8 (Fig. 9). Furthermore, aeration of the culture had a significant impact on enzyme production. The enzyme yield of the culture incubated under shaking conditions (150 rpm) was about 4 folds compared to static conditions, 40 and 11.8 U/mL, respectively (Fig. 10).
Partial purification and characterization of the enzyme
The partial purification of the protease enzyme was carried out with Sephadex G-100 column chromatography. The activity of the different fraction per mg of protein was determined. The fraction with the highest activity of protease was chosen for further characterization.
The partially purified protease was analyzed by zymography. Two clear bands showed casein degradation activities, at approximately 250 and 190 KDa (Fig. 11).
Effect of salts on protease activity
The effect of salts on HAL1 protease activity was studied using increasing NaCl (0 to 4 M) and KCl (0 to 3 M) concentrations. As shown in Fig. 12, the maximum relative activity of the enzyme was obtained at 0.5 M NaCl (106.5 ± 2.5). Beyond 0.5 M NaCl, enzyme activity decreased progressively, and only 40% of its relative activity remained at 4 M NaCl. However, the presence of up to 3 M KCl enhanced the enzyme activity, with maximum relative activity at 2 M KCl (163.3 ± 1.5).
Effect of temperature on protease activity
The effect of different temperatures (5–80 °C) on enzyme activity was investigated. According to our findings, the activity of the enzyme peaked at 50 °C and remained active up to 80 °C (Fig. 13).
Effect of pH on protease activity
The effect of pH on HAL1 protease was studied at different pH (4-11) at 50 °C using casein as substrate. The enzyme was significantly active between pH 7 and 11 with optimum activity (508.1 U/mg protein) at pH 9 (Fig. 14). The enzyme showed about 2.6 and 5.9% activity reduction at pH 10 and 11, respectively.
Effect of organic solvents on protease activity
Table 2 summarizes the impact of some polar and non-polar organic solvents (− 0.24 ≤ log Po/w ≤ 3.5), at 25% concentration, on HAL1 protease activity. The inclusion of hydrophobic solvents with a partition coefficient in the octanol/water two-phase (log Po/w) greater than 3 increased the activity of the enzyme. Organic solvents with log Po/w ≤ 2.13, on the other hand, reduced the protease activity.
Effect of metal ions and EDTA on the activity of HAL1 protease
Table 3 shows the effect of some divalent metal ions on the activity of HAL1 protease. According to our findings, the presence of Ca2+, Mg2+, and Mn2+ increased the activity of the enzyme. The presence of 10 mM Mn2+ ions resulted in the greatest increase in enzyme activity (1165.8% ± 4.7). Pb2+ ions had a minor inhibitory effect at 5 and 10 mM, while Zn2+ had the strongest inhibitory effect at 10 mM. The effect of different EDTA concentration (5, 10, and 15 mM) on HAL1 protease activity was studied. The enzyme activity was increased with increasing EDTA concentration. The respective relative activities were 118.2, 213.6, and 315% (Fig. 15).
Effect of surfactants and H2O2 on the activity of HAL1 protease
The effect of surfactants and H2O2 on the activity of HAL1 protease was studied, and it was found that incubation with Tween-80, SDS, and H2O2 (1, 5, and 10%) stimulated the activity to varying degrees, with H2O2 having the highest stimulation effect at 10% concentration (227.4% ± 1.0). The presence of 1 and 5% concentrations of Triton-X100 caused a minor change in enzyme activity. However, the enzyme activity decreased sharply to 55.2% at 10% concentration of Triton-X100 (Table 4).
We isolated six bacterial isolates from saline soil, and one isolate, coded as strain HAL1, was chosen for its ability to produce alkaline protease. The strain was identified as a species of the genus Halobacillus and designated as Halobacillus sp. HAL1 with the GenBank accession number OK001470. In addition, we carried out further investigations concerning optimization of production, partial purification, and characterization of the protease produced by the strain. Our findings revealed that the enzyme has a high molecular weight and is compatible with surfactants, EDTA, metal ions, and organic solvents, indicating that it is suitable for a variety of industrial applications.
Thalssohaline environments are hypersaline environments that originate from the sea and contain salts that have an ionic composition similar to seawater. However, the concentration of seawater (3.5% salinity, on average) due to solar evaporation causes serial precipitation of various salts including, calcium carbonate, sodium chloride, and the salts of Mg+2 and K+ ions. These habitats are either naturally occurring or man-made salterns. Sabkhas, also known as saline soil or evaporites, are good examples of the natural hypersaline environments . Only halophilic and halotolerant microorganisms thrive in these environments, which have lower microbial diversity than seawater . The extreme saline conditions in these environments favors the growth of microbes possessing unique adaptive characteristics that could be exploited in a variety of biotechnological applications, particularly hydrolytic enzymes . In this study, we isolated six isolates of heterotrophic bacteria from saline soil and assessed their capacity to produce alkaline protease. One isolate, designated HAL1, had the highest alkaline protease activity and was chosen for further characterization and production of alkaline protease. The isolate is a Gram-positive rod that requires sodium for growth, and can tolerate up to 24% of NaCl with optimum growth achieved at 9% of NaCl and, therefore, was considered a moderately halophilic bacterium .
Phylogenetic analysis of the 16 s rDNA of the isolate confirmed the affiliation of the isolate to the genus Halobacillus and it was designated as Halobacillus sp. HAL1; the sequence was deposited in the GenBank under the accession number OK001470. Over the past decades, microorganisms inhabiting saline and hypersaline environments have been the subject of interest for the bioprospecting of valuable hydrolytic enzymes [38,39,40]. Several studies have reported the isolation of halotolerant and halophilic microbes producing potent hydrolases that are active under extreme conditions of salinity and pH, especially members of the genus Halobacillus [41,42,43,44,45].
The cost-effective production of enzymes represents a great challenge for the industrial application of enzymes. Therefore, the use of low-cost substrates is urgently needed to enhance the use of enzymes in various industries in an economical way . The utilization of fish waste substrate, which provides an excellent nutrient source for microbial growth and enzyme production, could solve this issue . In the present study, HAL1 produced higher protease activity when grown in media containing only fish waste substrate, and this could lower the cost of enzyme production.
The low yield of enzymes and other metabolites from extremophiles is one of the major obstacles in their industrial applications . The effects of culture media composition and the culturing conditions such as aeration level, temperature, pH, and incubation time on the production of alkaline protease have been confirmed [48,49,50]. To obtain a high yield of alkaline protease, it is critical to optimize the composition of the production medium and the culturing conditions. The optimal culturing conditions for alkaline protease production by HAL1 were studied, and the results revealed that the strain produces the highest yield of alkaline protease when cultured in an artificial seawater based medium containing 10 g/L of SC waste powder and 30 g/L of NaCl, pH 8 and incubated at 25 °C under shaking conditions (150 rpm). The ability of HAL1 to grow and produce protease in a medium containing only SC waste substrate alone indicates that this substrate could promote the growth and protease production without the need for additional nutrients. The requirement for alkaline pH for optimal protease production suggests the alkaliphilic nature of the strain and the produced protease [22, 51]. In addition, the enhanced protease production under shaking conditions indicates the aerobic nature of strain HAL1 .
Microbial proteases have molecular masses ranging from 15 to 40 kDa [16, 53]. Thus, Karbalaei-Heidari et al.  identified an extracellular alkaline protease from the moderately halophilic bacterium Halobacillus karajensis with molecular weight of 36 kDa, whereas alkaline protease from Halobacillus andaensis is about 18 kDa  and from Halobacillus sp. CJ4 of 18 to 30 kDa . Santos et al.  detected several proteases with molecular masses ranging from 30 to 80 kDa in Halobacillus blutaparonensis. However, Dorra et al.  identified a high molecular weight alkaline protease of about 250 kDa produced by Bacillus halotolerans strain CT2. The partially purified protease secreted by HAL1 showed two casein degradative activities with molecular masses of 190 and 250 kDa. To our knowledge, this is the first report on a high molecular weight protease from a Halobacillus species.
The enzymes of halophilic microorganisms are adapted to function in hypersaline environments and possess unique properties, including thermostability and pH tolerance. In addition, halophilic enzymes are resistant to denaturation and can catalyze in low water activity [59,60,61,62]. Among halophilic enzymes, proteases find a wide application in pharmaceuticals, leather tanning, food, and detergent industries due to its stability under harsh industrial conditions . The effects of salts (NaCl and KCl) and pH on the activity of HAL1 protease were studied, and our results revealed that 0.5 M NaCl, 2 M KCl, and pH 9 are the optimum conditions for maximum activity of the enzyme. These results indicate the halo-alkaline nature of the enzyme and are similar to the optimal conditions for proteases from Halobacillus sp. CJ4 strains  and Halobacillus. karajensis MA-2 . Because HAL1 protease showed excellent thermostability at wide range of temperatures, from 30 to 80 °C, with an optimum temperature of 50 °C, it was considered a thermostable enzyme. a Similar temperature optimum was reported for alkaline protease from Halobacillus karajensis MA-2  and Bacillus mojavensis .
Besides thermal stability and activity at high pH, proteases that are stable in the presence of organic solvents, oxidizing agents, metal ions, and surfactants are attractive for industrial applications . Protease-catalyzed reactions are often carried out in non-aqueous media, so proteases that are stable in the presence of organic solvents would be valuable for synthesis in such environments . According to Laane et al. , the logarithm of the partition coefficient of the solvent between octanol and water (log Po/w) is the best parameter for relating the enzyme activity to the solvent nature. Thus, hydrophobic solvents (having high log Po/w values) cause less inactivation of biocatalysts than solvents with lower log Po/w values. The presence of hexane and xylene (log Po/w greater than 3) induced the activity of HAL1 protease, whereas organic solvents having log Po/w less than 0 or equal to 2.1 deactivated the enzyme by about 20%. Butanol (log Po/w, 0.8) deactivated HAL1 protease by 84%. Hydrophilic solvents destabilize enzymes by removing the water hydration shell of the enzyme which is essential for structure flexibility and catalytic activity , and this could account for the decrease in HAL1 protease activity in the presence of hydrophilic solvents.
The effect of some divalent metal ions on the activity of HAL1 protease was studied. Among the tested metal ions, the presence of Ca 2+, Mn 2+, and Mg 2+ (5 and 10 mM) enhanced the enzyme activity; the highest activation effect was observed in the presence of 10 mM Mn2+ ions (1165% relative activity). Similarly, Ca2+ and Mg2+ ions have previously been shown to activate protease enzyme [67,68,69,70,71,72,73,74]. Yu et al.  found that Mn2+ ions (10 mM) had a lower activation effect on alkaline protease from Bacillus sp. ZJ1502, compared with the effect on HAL1 protease (122 and 1165% relative activity; respectively). The significant enhancement of the enzyme caused by the addition of Mn2+ ions suggests that this metal ion facilitates the binding of the substrate to the active site of the enzyme . On the other hand, Zn2+ and Fe2+ ions partially inhibited the enzyme; similar inhibition effects have been reported for alkaline protease from Bacillus sp. ZJ1502  and Bacillus halotolerans strain CT2 . Interestingly, HAL1 protease was stable in the presence of Pb2+ ions (5 and 10 mM) but lost about 70% of its activity in the presence of Cd2+ ions. Some metal ions may inhibit protease activity by binding to specific amino acids that are important for catalytic function or by affecting the charge distribution of the enzyme molecules . The activity of HAL1 protease was increased in the presence of EDTA (5, 10, and 15 mM), and the relative activities were 118.2, 213.6, and 315%, respectively. The significant increase of enzyme activity in the presence of EDTA is a novel finding and suggests the enzyme is not a metalloprotease enzyme  and is suitable for the detergent industry, particularly because chelating agents such as EDTA are commonly used in detergent formulation [68, 71].
Surfactants and oxidizing agents are commonly used in the formulation of modern detergents. Therefore, alkaline proteases that are stable in the presence of oxidizing agents and surfactants are crucial in the detergent industry . Incorporation of Tween 80, SDS, or H2O2 (up to 10%) and Triton-X 100 (up to 5%) into the reaction mixture enhanced the activity of HAL1 protease; the relative activity in the presence of 10% H2O2 was 227.1%. Similar activation effect of Tween 80 and Triton- × 100 have been reported previously for alkaline protease from Bacillus invictae , while a higher activation effect was observed for serine alkaline protease from Bacillus safensis strain RH12  and crude protease from Bacillus cereus SV1 . Lower activation effect of oxidizing agents for other alkaline proteases have been reported [68, 75, 79,80,81,82,83]. However, protease from Aeribacillus pallidus C10 showed weak stability in the presence of H2O2 . In comparison to all of these proteases, HAL1 protease showed a high level of oxidizing agents compatibility. In addition, stability of HAL1 protease was improved in the presence of up to 10% SDS; few studies have reported the stability of proteases in the presence of SDS .
In the current study, the moderately halophilic bacterium, Halobacillus strain HAL1 was isolated from saline soil and used to produce an extracellular alkaline protease using fish waste substrate as the sole nutritional source. The enzyme secreted by HAL1 strain was found to be a novel high molecular weight alkaline protease with molecular masses of 190 and 250 kDa and to exhibit novel properties that make it suitable for detergent formulations such as alkaline pH and thermal stability, as well as high compatibility with metal ions, organic solvents, surfactants, EDTA, and H2O2. Further research is needed to fully understand its structure organization, as well as the possible industrial applications.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Compound annual growth rate
Fish processing waste
Polymerase chain reaction
Basic local alignment search tools
Sodium dodecyl sulphate
Liu L, Yang H, Shin HD (2013) How to achieve high-level expression of microbial enzymes strategies and perspectives. Bioengineered 4(4):212–223
Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. Biomed Res Int 329121:18
Singh R, Kumar M, Mittal A, Mehta PK (2016a) Microbial enzymes: industrial progress in 21st century. 3 Biotech 6:174
Adrio JL, Demain AL (2014) Microbial enzymes: tools for biotechnological processes. Biomolecules 4:117–139
Nath IVA, Bharathi PAL (2011) Diversity in transcripts and translational pattern of stress proteins in marine extremophiles. Extremophiles 15:129–153
Di Donato P, Buono A, Poli A, Finore I, Abbamondi RG, Nicolaus B, Lama L (2018) Exploring marine environments for the identification of extremophiles and their enzymes for sustainable and green bioprocesses. Sustainability 11:149
Zhu D, Adebisi WA, Ahmad F, Sethupathy S, Danso B, Sun J (2020) Recent development of extremophilic bacteria and their application in biorefinery. Front Bioeng Biotechnol 8:483
Delgado-García M, Valdivia-Urdiales B, Aguilar-Gonzalez CN, Contreras-Esquivel JC, Rodriguez-Herrera R (2012) Halophilic hydrolases as a new tool for the biotechnological industries. J Sci Food Agric 92(13):2575–2580
De Lourdes MM, Pérez D, García MT, Mellado E (2013) Halophilic bacteria as a source of novel hydrolytic enzymes. Life 3(1):38–51
Liu C, Baffoe DK, Zhang M (2018) Halophile, an essential platform for bioproduction. J Microbiol Methods 6:105704
Raveendran S, Parameswaran B, Ummalyma SB, Abraham A, Mathew AK, Madhavan A, Rebello S, Pandey A (2018) Applications of microbial enzymes in food industry. Food Technol Biotechnol 56(1):16–30
Beg QK, Gupta R (2003) Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme Microb Technol 32:294–304
Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, Ashraf M (2019) Microbial Proteases Applications. Front. Bioeng. Biotechnol 7:110
Sharma KM, Kumar R, Panwar S, Kumar A (2017) Microbial alkaline proteases: optimization of production parameters and their properties. J Genet Eng Biotechnol 15:115–126
Hammami A, Hamdi M, Abdelhedi O, Jridi M, Nasri M, Bayoudh A (2017) Surfactant- and oxidant-stable alkaline proteases from Bacillus invictae: Characterization and potential applications in chitin extraction and as a detergent additive. Int J Biol Macromol 96:272–281
Gupta R, Beg QK, Khan S, Chauhan B (2002) An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol 60:381–395
Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–32
Rebah FB, Miled N (2013) Fish processing wastes for microbial enzyme production: a review. 3 Biotech 3:255–265
Taher AG (2014) Microbially induced sedimentary structures in evaporite–siliciclastic sediments of Ras Gemsa sabkha, Red Sea Coast. Egypt J Adv Res 5:577–586
Albokari MA, Cinar S, Mutlu MB (2017) Microbial characterization of jazan sabkha in saudi arabia. Appl Ecol Environ Res 15(3):1069–1077
Dang H, Zhu H, Wang J, Li T (2009) Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough. World J Microbiol Biotechnol 25:71–79
Horikoshi K (1999) Alkaliphiles: some applications of their products for biotechnology. Microbiol Mol Biol Rev 63:735
Karray F, Abdallah MB, Kallel N, Hamza M, Fakhfakh M, Sayadi S (2018) Extracellular hydrolytic enzymes produced by halophilic bacteria and archaea isolated from hypersaline lake. Mol Biol Rep 45:1297–1309
Bergey DH, Buchanan RE, Gibbons NE (1974) Bergey’sManual of Determinative Bacteriology, 8th edn. Williamsand Wilkins ce, Baltimore
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30(5):1229–1235
Kalwasińska A, Jankiewicz U, Felföldi T, Burkowska-But A, Brzezinska MS (2018) Alkaline and halophilic protease production by Bacillus luteus H11 and its potential industrial applications. Food Technol Biotechnol 56(4):553
Ellouz Y, Bayoudh A, Kammoun S, Gharsallah N, Nasri M (2001) Production of protease by Bacillus subtilis grown on sardinelle heads and viscera flour. Bioresour Technol 80:49–51
Garcı´a-Carren OF, Haard N, Dimes N (1993) Substrategel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal Biochem 214:65–69
Moshfegh M, Shahverdi AR, Zarrini G, Faramarzi MA (2013) Biochemical characterization of an extracellular polyextremophilic a-amylase from the halophilic archaeon Halorubrum xinjiangense. Extremophiles 17:677–687
Sanatan PT, Lomate PR, Giri AP, Hivrale VK (2013) Characterization of a chemostable serine alkaline protease from Periplaneta americana. BMC Biochem 14:32
Amoozegar MA, Malekzadeh F, Malik KA, Schumann P et al (2003) Halobacillus karajensis sp. nov., a novel moderate halophile. Int J Sys Evol Microbiol 53:1059–1063
Spring S, Ludwig W, Marquez MC, Ventosa A et al (1996) Halobacillus gen. nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov. Int J Syst Bacteriol 46:492–496
Ventosa A, Arahal DR (2009) Physico-chemical characteristics of hypersaline environments and their biodiversity. Extremophiles 2:1–6
Safarpour A, Amoozegar MA, Ventosa A (2018) Hypersaline environments of Iran: Prokaryotic biodiversity and their potentials in microbial biotechnology. In: Egamberdieva D, Birkeland NK, Panosyan H, Li WJ (eds) Extremophiles in Eurasian ecosystems: ecology, diversity, and applications. Springer, Singapore, pp 265–98 (Volume 8)
Ghosh S S, Kumar S S, Khare SK SK (2019) Microbial diversity of saline habitats: an overview of biotechnological applications. In: Giri B, Varma A (eds) Microorganisms in saline environments: Strategies and Functions. Springer, Cham, pp 65–92. (ISBN-13: 978–3–030–18975–4)
Kushner DJ (1993) Growth and nutrition of halophilic bacteria. In: Vreeland RH, Hochstein L (eds) The biology of halophilic bacteria. CRC, Boca Raton, FL, pp 87–103
Ventosa A, Nieto JJ (1995) Biotechnological applications and potentialities of halophilic microorganisms. World J Microbiol Biotechnol 11:85–94
Oren A (2010) Industrial and environmental applications of halophilic microorganisms. Environ Technol 31:825–834
Liu C, Baffoe DK, Zhan Y, Zhang M, Li Y, Zhang G (2019) Halophile, an essential platform for bioproduction. J. Microbiol. Methods 166:105704
Sánchez-Porro C, Martín S, Mellado E, Ventosa A (2003) Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes. J Appl Microbiol 94:295–300
Rohban R, Amoozegar MA, Ventosa A (2009) Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake. Iran J Ind Microbiol Biotechnol 36(3):333–340
Menasria T, Aguilera M, Hocine H, Benammar L, Ayachi A, Si Bachir A, Dekak A, Monteoliva-Sánchez M (2018) Diversity and bioprospecting of extremely halophilic archaea isolated from Algerian arid and semi-arid wetland ecosystems for halophilic-active hydrolytic enzymes. Microbiol Res 207:289–298
Amoozegar MA, Safarpour A, Noghabi KA, Bakhtiary T, Ventosa A (2019) Halophiles and their vast potential in biofuel production. Front Microbiol 10:1895
Kaitouni LBD, Anissi J, Sendide K, El Hassouni M (2020) Diversity of hydrolase-producing halophilic bacteria and evaluation of their enzymatic activities in submerged cultures. Ann Microbiol 70:33
Sakhuja D, Ghai H, Rathour RK, Kumar P, Bhatt AK, Bhatia RK (2021) Cost-effective production of biocatalysts using inexpensive plant biomass: a review. 3 Biotech 11:280
Joshi RH, Dodia MS, Singh SP (2008) Production and optimization of a commercially viable alkaline protease from a haloalkaliphilic bacterium. Biotechnol Bioprocess Eng 13:552–559
Patel RK, Dodia MS, Joshi RH, Singh SP (2006) Production of extracellular halo-alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. isolated from seawater in Western India. World J Microbiol Biotechnol 22:375–382
Deng A, Wu J, Zhang Y, Zhang G, Wen T (2010) Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour Technol 101:7100–7106
Patel R, Dodia M, Singh SP (2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: production and optimization. Process Biochem 40:3569–3575
Horikoshi K, Antranikian G, Bull AT, Robb FT, Stetter KO (2011) Extremophiles Handbook. Springer, Berlin vol. 1 – 2
Ibrahim ASS, Al-Salamah AA, Elbadawi YB, El-Tayeb MA, Ibrahim SSS (2015) Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes. Electron J Biotechnol 18:236–243
Ibrahim ASS, Al-Salamah AA, El-Badawi YB, El-Tayeb MA, Antranikian G (2015) Detergent, solvent and salt compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization. Extremophiles 19:961–971
Karbalaei-Heidari HR, Amoozegar MA, Hajighasemi M, Ziaee AA, Ventosa A (2009) Production, optimization and purification of a novel extracellular protease from the moderately halophilic bacterium Halobacillus karajensis. Ind Microbiol Biotechnol 36:21–27
Delgado-García M, Flores-Gallegos AC, Kirchmayr M, Rodríguez JA, Mateos-Díaz JC, Aguilar CN, Muller M, Camacho-Ruíz RM (2019) Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein. Electron J Biotechnol 39:52–60
Daoud L, Jlidi M, Hmani H, Brahim A, El Arbi M, Ben Ali M (2017) Characterization of thermo-solvent stable protease from Halobacillus sp. CJ4 isolated from Chott Eldjerid hypersaline lake in Tunisia. J Basic Microbiol 57:104–113
Santos AF, Valle RS, Pacheco CA, Alvarez VM, Seldin L, Santos ALS (2013) Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium. Braz J Microbiol 44(4):1299–1304
Dorra G, Ines K, Imen BS, Laurent C, Sana A, Tabbene O, Pascal C, Thierry J, Ferid L (2018) Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2. Int J Biol Macromol 111:342–351
Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4(2):91–98
Karan R, Kumar S, Sinha R, Khare SK (2012) Halophilic microorganisms as sources of novel enzymes. In: Satyanarayana T, Johri BN, Prakash A (eds) Microorganisms in sustainable agriculture and biotechnology. Springer, Dordrecht, pp 555–579
Sinha R, Khare SK (2014) Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation. Front Microbiol 5:165
Cira-Chávez LA et al (2018) Kinetics of halophilic enzymes. In: Rajendran L, Fernandez C (eds) Kinetics of Enzymatic Synthesis. IntechOpen, London. https://doi.org/10.5772/intechopen.81100
Lakshmi BKM, Kumar DM, Hemalatha KPJ (2018) Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8. J Genet Eng Biotechnol 16:295–304
Ogino H, Yasui K, Shiotani T, Ishihara T, Ishikawa H (1995) Organic solvent stable-tolerant bacterium which a secretes an organic solvent-stable proteolytic enzyme. Appl Environ Microbiol 61(12):4258–4262
Laane C, Boeren S, Vos K, Veeger C (1987) Rules for optimization of biocatalysis in organic solvents Biotechnol. Bioeng 30:81–87
Ebrahimpour A, Rahman RN, Basri M, Salleh AB (2011) High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp strain ARM. Bioresour Technol 102:6972–6981
Gerday C, Aittaleb M, Bentahir M et al (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107
Haddar A, Bougatef A, Agrebi R et al (2009) A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21 Purification and characterization. Process Biochem 44:29–35
Rao S, Sathish T, Ravichandra P, Prakasham R (2009) Characterization of thermo- and detergent stable serine protease from isolated Bacillus circulans and evaluation of ecofriendly applications. Process Biochem 44:262–268
Annamalai N, Rajeswari MV, Thavasi R et al (2013) Optimization, purification and characterization of novel thermostable, haloalkaline, solvent stable protease from B. halodurans CAS6 using marine shellfish wastes: a potential additive for detergent and antioxidant synthesis. Bioprocess Biosyst Eng 36:873–883
Patil U, Mokashe N, Chaudhari A (2016) Detergent-compatible, organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: purification and characterization. Prep Biochem Biotechnol 46:56–64
Iqbal A, Hakim A, Hossain MS, Rahman MR, Islam K, Azim MF et al (2018) Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2. J Gen Eng Biotechnol 16:29–37
Patel AR, Mokashe NU, Chaudhari DS, Jadhav AG, Patil UK (2019) Production, optimisation and characterisation of extracellular protease secreted by newly isolated Bacillus subtilis AU-2 strain obtained from Tribolium castaneum gut. Biocatal Agric Biotechnol 19:101–122
Zhang J, Wang J, Zhao Y, Li J, Liu Y (2019) Study on the interaction between calcium ions and alkaline protease of Bacillus. Int J Biol Macromol 124:121–130
Yu P, Huang X, Ren Q, Wang X (2019) Purification and characterization of a H2O2-tolerant alkaline protease from Bacillus sp. ZJ1502, a newly isolated strain from fermented bean curd. Food Chem 274:510–517
Li F, Yang L, Lv X, Liu D, Xia H, Chen S (2016) Purification and characterization of a novel extracellular alkaline protease from Cellulomonas bogoriensis, Protein Expr. Purif 121:125–132
Ekhlas M, Rahman UM, Faruquee HM, Islam Khan MR, Mortuza MF, Rahman MH, Maitra P (2015) Isolation, identification and partial characterization of protease producing bacteria that exhibiting remarkable dehairing capabilities. Glob Jou Inc C Biol Sci 15:2249–4626
Rekik H, Jaouadi NZ, Gargouri F, Bejar W, Frikha F, Jmal N et al (2019) Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12. Int J Biol Macromol 121:1227–1239
Manni L, Ghorbel-Bellaaj O, Jellouli K, Younes I, Nasri M (2009) Extraction and characterization of chitin, chitosan, and protein hydrolysates prepared from shrimp waste by treatment with crude protease from Bacillus cereus SV1. Appl Biochem Biotechnol 162:345–357
Manni L, Misbah A, Zouine N, Ananou S (2020) Biochemical characterization of a novel alkaline and detergent stable protease from Aeromonas veronii OB3. Microbiol Biotechnol Lett 48(3):358–365
Biver S, Portetelle D, Vandenbol M (2013) Characterization of a new oxidant-stable serine protease isolated by functional metagenomics. Springerplus 2:410
Sellami-Kamoun A, Haddar A, Ali NEH, Ghorbel-Frikha B, Kanoun S, Nasri M (2008) Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations Microbiol. Res 163:299–306
Divakar K, Deepa Arul Priya J, Gautam P (2010) Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01. J Mol Catal B: Enz 66:311–318
Yildirim V, Baltaci MO, Ozgencli I, Sisecioglu M, Adiguzel A, Adiguzel G (2017) Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents. J Enzyme Inhib Med Chem 32(1):468–477
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Fahmy, N.M., El-Deeb, B. Optimization, partial purification, and characterization of a novel high molecular weight alkaline protease produced by Halobacillus sp. HAL1 using fish wastes as a substrate. J Genet Eng Biotechnol 21, 48 (2023). https://doi.org/10.1186/s43141-023-00509-6
- Fish processing wastes
- Alkaline protease
- Saline soil