Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30

Abstract

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80℃ in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50℃ and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni²⁺, Mn²⁺, and Cu²⁺ to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The K_m and V_max values were estimated to be 35.7 mg/ml, and 5 × 10⁴ U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

Introduction

Alkaline proteases (E.C. 3.4.21) are a major group of industrial enzymes that hydrolyze proteins into short peptides and amino acids, and catalyze peptide synthesis [7,8]. Proteases account for about 60% of the total worldwide enzyme sales [26], as they are widely used in the production of detergents, food, leather goods, pharmaceuticals, and textiles [28]. There is a significant amount of interest, from a biotechnological perspective, in alkaline proteases produced by microorganisms [36]. Most alkaline proteases, such as the commercial proteases Subtilisin Carlsbery, Alcalase, Esparase, and Savinase, are produced by bacteria, in particular, Bacillus sp. [5,14,34]. The proteases produced from actinomycetes have received less attention despite reports that they display unique characteristics [31,33]. The production of novel proteases from actinomycetes should be investigated so as to meet the requirements of different industrial processes [9].

In this study, Streptomyces sp. M30 was isolated and identified. A novel extracellular alkaline protease was purified and characterized from strain M30 by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography. The purified protease exhibited high catalytic activity and stability under different extreme conditions. SapHM was highly stable in the presence of various surfactants; therefore, it could be considered an appropriate detergent additive.

 

Materials and Methods

Chemicals

Casein, Folin’s phenol, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO, USA). DEAE-Sepharose chromatography and hydrophobic interaction chromatography columns were purchased from GE-Healthcare Life Sciences (UK). All other chemicals used in this study were of analytical grade or higher purity.

Isolation and Identification of Protease-Producting Strain

A species of Streptomyces sp. that was able to produce protease was isolated from soil samples (Nanjing, China) using the plate dilution technique on Gause’s agar medium (8 g/l soluble starch, 1.7 g/l KCl, 0.5 g/l KNO3, 0.5 g/l Na2HPO4·12H2O, 0.5 g/l MgSO4·7H2O, 0.02 g/l CaCO3, 0.01 g/l FeSO4) supplemented with 20 mg/ml casein. All plates were incubated at 28℃ and the different colonies of Streptomyces sp. associated with transparent halos indicative of casein hydrolysis were isolated. The protease activity of the isolates was identified again according to the diameter of the transparent halo. The strain producing maximum alkaline protease was identified on the basis of its 16S rRNA gene sequence and named as Streptomyces sp. M30. The phylogenetic tree was constructed using MEGA5 software and the sequence was submitted to the GenBank database. Distances were calculated using the Kimura two-parameter distance model. Phylogenetic trees were generated using the neighbor-joining method and evaluated by bootstrap analyses based on 1,000 resamplings.

Streptomyces sp. M30 was cultured in 250 ml Erlenmeyer flasks containing 100 ml of Gause’s liquid medium at 28℃ and 180 rpm. These cells were then used to inoculate (1% inoculum) a larger volume of fresh Gause’s liquid medium, and incubated at 28℃ and 180 rpm. Protease activity was monitored at on interval of 24 h, as described in the following section. The optimal protease-producing time of Streptomyces sp. M30 could be judged according to the measured results.

Protease Activity Assay

Protease activity was determined using Folin’s phenol [21]. First, 4 μl of enzyme was added to 2 ml of 50 mM buffer A (glycine-NaOH, pH 9.0) containing 20 mg/ml casein, and the mixtures were incubated at 75℃ for 10 min. Reactions were terminated by adding 2 ml of 0.4 M trichloroacetic acid and incubating at 75℃ for 20 min. The mixtures were centrifuged (12,000 rpm, 5 min), and the supernatants were mixed with 5 ml of 0.4 M sodium carbonate buffer and 1 ml of Folin’s phenol, and then incubated at 37℃ for 20 min. The absorbance of each sample was measured at 660 nm. A single unit of protease activity was defined as the amount of enzyme that released 1 μg of tyrosine per minute under optimum assay conditions.

Protease activity in the presence of casein (0.8-5.0 mg/ml) was evaluated in 50 mM buffer A at 75℃. The kinetic parameters (Michaelis-Menten constant, Km, and maximal reaction velocity, Vmax) were calculated using Lineweaver-Burk double-reciprocal plots [11].

Purification of Alkaline Protease SapHM and Zymogram Analysis

Two liters of fermentation supernatant of strain M30 was harvested as the crude enzyme preparation of protease by centrifugation (10,000 rpm, 10 min, 4℃). Ammonium sulfate was gradually added to the supernatants until the concentration reached 100% and precipitates were collected by centrifugation (12,000 rpm, 20 min, 4℃). The precipitates were then dissolved in 20 ml of 40% ammonium sulfate, which was prepared with 50 mM buffer A. Protein preparations were then purified by hydrophobic interaction chromatography (1 × 10 cm); bound proteins were eluted with 40%-0 linear concentration gradient of ammonium sulfate at a flow rate of 0.5 ml/min, with 0.5 ml fractions collected. Fractions containing protein were pooled and dialyzed against 50 mM buffer A overnight with frequent buffer changes. The dialyzed protein suspension was then subjected to DEAE-Sepharose chromatography (1.0 × 10 cm). The bound proteins were eluted with a linear gradient of NaCl from 0 to 1 M at a flow rate of 0.5 ml/min, with 1.0 ml fractions collected. The fractions containing protease were collected and concentrated.

The concentration of the purified protease was determined according to the Bradford method [6]. The purity and molecular mass of the purified protease were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and MS, as described by Laemmli [19]. MS (Bruker Daltonics) analysis was performed at Zoonbio Biotechnology Co., Ltd (Nanjing, China).

SDS-PAGE gels were cut into two strips for staining and zymogram analysis [20]. One strip was stained with 1 mg/ml Coomassie Brilliant Blue R250 in 10% (v/v) acetic acid and 45% (v/v) ethanol. Destaining was performed with 5% (v/v) ethanol and 7.5% (v/v) acetic acid. The other gel strip was incubated in 2.5% Triton X-100 to remove SDS (2 × 30 min). The strip was then incubated in 0.1 M glycine-NaOH (pH 8.0, 2 × 30 min), and then in 50 mM buffer A (2 × 15 min) to renature the enzyme. The gel strip was then placed on a 15 mg/ml agar plate containing 20 mg/ml casein for 10 min at 50℃, until a clear transparent halo appeared on the plate. The two gel strips were compared, and the band on the Coomassie-stained gel corresponding to the position of the transparent halo was excised. Gel slices were then stored at -80℃ for peptide mass fingerprint analysis.

Protein Sequencing Analysis and Gene Cloning

According to the result of zymogram analysis, gel slices containing the protease were identified by peptide mass fingerprinting (Bo-Yuan Biological Technology Co. Ltd., Shanghai, China). The resulting peptide fragments were analyzed by searching Mascot databases (http://www.matrixscience.com) to determine the sequences of peptide fragments. The corresponding protein was searched against the GenBank database using the BLASTP program (http://blast.ncbi.nlm.nih.gov). In addition, a phylogenetic tree was generated based on the amino acid sequence by MEGA5 software. The putative signal peptide of protease was predicted using the SignalP server (http://www.cbs.dtu.dk/services/SignalP).

Genomic DNA of Streptomyces sp. M30 was extracted to serve as the polymerase chain reaction (PCR) template [13]. The protease gene was amplified using a pair of primers, sapHMF (5’-ATGCGCAAGACAGTTCTTGC-3’) and sapHMR (5’-TTACGGGACCTGGAGGAGCT-3’), which were designed by Primer 5.0 software based on nucleotide sequence. The product of PCR was inserted into pMD19-T to obtain the plasmid pMD19T-sapHM and sequenced by Invitrogen Co. Ltd (Shanghai, China).

Biochemical Characterization of SapHM

The substrate specificity of the purified enzyme was determined using 10 mg/ml casein, bovine serum albumin (BSA), bovine serum fibrin (BSF), and gelatin [18]. Substrates were prepared in 50 mM buffer A. Protease activity was assayed as described above.

Effects of temperature and pH on the protease activity and stability. The optimum temperature for enzyme activity was assessed from 20 to 90℃. The thermal stability of the protease was evaluated by incubating the enzyme at different temperatures (20-80℃) for 10-150 min in glycine-NaOH buffer (pH 9.0). Residual protease activity was measured using the enzyme assay described above. The activity of the enzyme before incubation was assumed to be 100%. The optimum pH for protease activity was assessed at 75℃ using the following buffers: 50 mM citrate buffer, pH 3.0-6.0; 50 mM phosphate-buffered saline, pH 6.0-8.0; and 50 mM glycine-NaOH buffer, pH 8.0-11.0. The pH stability of the protease was determined by incubating the enzyme at 4℃ for 24 h in buffers of varying pH. Residual enzyme activity was determined under optimum conditions.

Effects of metal ions and chemicals on enzyme activity. The effects of metal ions and chemicals on the activity of SapHM was determined at 75℃ for 10 min. The effects of various metal ions (Cu2+, K+, Co2+, Ni2+, Fe3+, Zn2+, Ca2+, Mg2+, Cr3+, Ba2+, and Mn2+), inhibitors (PMSF; urea; ethylenediaminetetraaceticacid (EDTA); ethylene glycol tetraacetic acid (EGTA); and dithiothreitol (DTT)), solvents (methanol, ethanol, acetone, acetonitrile, isopropanol, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO)), and surfactants (Tween 80, TritonX-100, SDS, and hexadecyl trimethyl ammonium bromide (CTAB)) on protease activity were investigated. The residual activity was determined as mentioned above. The effects of metal ions were determined after dialysis of the enzyme against 50 mM glycine-NaOH (pH 9.0). The enzyme activity was assumed to be 100% when additives were absent.

All determinations were performed in three replicates, and the control experiment without protease was carried out under the same conditions. The experimental results were expressed as the mean of the replicate determinations and standard deviation (SD). Statistical significance was evaluated using t-tests for two-sample comparison and one-way analysis of variance (ANOVA) followed by t-test. Statistical significance was defined by p < 0.05. R Version 3.1.1 (Vanderbilt University, USA) was used for the statistical analysis.

Nucleotide Sequence Accession Number

The nucleotide sequences of the Streptomyces sp. M30 16S rDNA gene and sapHM gene have been deposited in the GenBank database under the accession numbers KP408148 and KM885284, respectively.

 

Results

Isolation and Identification of Protease-Producting Strain

In the process of isolation and identification, colonies with different morphology were observed on agar plates. The colonies with zone of hydrolysis were designated as protease-producing strains. Three different colonies associated with casein hydrolysis were isolated and strain M30 showed the largest zone of casein hydrolysis with a diameter of 4.2 cm. The strain M30 colonies were opaque, dry, and exceedingly dense. Phylogenetic analysis of 16S rRNA gene confirmed the isolation as Streptomyces sp. (Fig. 1). It was named as Streptomyces sp. M30 and used as the experimental strain for further study. The 16S rRNA gene sequence has been deposited in the GenBank database.

Fig. 1.Multiple sequence alignment.

The protease yield curve of strain M30 was determined in order to purify the protease. Protease activity was not detected in the first 3 days, as the growth rate of strain M30 was relatively slow. However, it increased rapidly and was detected after 3 days. In addition, the growth rate of protease activity diminished to 7 days, peaking at 313 U/ml after 9 days. Moreover, the protease activity was stable and did not decrease after 9 days (Fig. 2). After cultivation, Streptomyces sp. M30 will become granular and the absorbance at 600 nm cannot be determined accurately. Thus, only biomass can be used to determine the growth of cells. The wet weight of cells was up to 5 g/l after 9 days, indicating that protease activity increased in parallel with the growth of Streptomyces sp. M30.

Fig. 2.Yield curve of alkaline protease from Streptomyces sp. M30.

Purification of Alkaline Protease SapHM

In order to obtain the biochemical characterization of SapHM, the protease from Streptomyces sp. M30 was purified by a combination of several steps. A summary of the purification process for protease is shown in Table 1. After the three-step purification process, there was a 7.0-fold increase in purification of protease, with a 15.5% recovery. The purified enzyme, which was designated as SapHM, was evident as a single band following SDS-PAGE (Fig. 3A). The molecular weight of SapHM cannot be obtained from SDS-PAGE because the migration rate of protein molecular weight standard was affected significantly. However, the result of mass spectrometry indicated that the molecular mass of SapHM was 37.1 kDa, as showed in Fig. 4.

Table 1.Purification procedure for SapHM.

Fig. 3.Purification and zymogram analysis of SapHM.

Fig. 4.Molecular mass of SapHM based on mass spectrometry analysis.

Biochemical Characterization of SapHM

The effects of temperature and pH on the protease activity of SapHM were assayed using purified enzyme. SapHM exhibited highest activity at 80℃ (Fig. 5A), with relatively high activity over 70-85℃. We also found that SapHM was stable from 20 to 40℃, and retained 65% of its initial activity after 2-h incubation at 50℃ (Fig. 5B). However, incubation at 60℃ and 70℃ for 30 min resulted in a decrease of activity to 40% and 0%, respectively. The stability of SapHM at 80℃ was poor, whereas it displayed good activity and stability following incubation at 75℃ for 10 min; therefore, the optimal temperature of reactions was set at 75℃.

Fig. 5.Effects of temperature and pH on SapHM activity and stability.

The activity of SapHM was observed over a broad pH range of 6.0-11.0, with maximum activity at pH 9.0. A sharp decrease in activity was observed at pH 5.0 (Fig. 5C). The activity of SapHM was stable over a pH range of 4.0-10.0, with 90% activity between pH 6.0 and 10.0 (Fig. 5D).

Metal ions play an important role in the activity of enzymes. The effects of various metal ions on SapHM activity are summarized in Table 2. The enzyme retained over 90% of its activity in the presence of Ba2+, Cr3+, K+, Fe3+, Ca2+, Zn2+, and Mg2+ at a concentration of 1 mM. Enzyme activity was significantly increased in the presence of Cu2+, Mn2+, and Ni2+ to 147%, 113%, and 112% of control, respectively.

Table 2.Effects of metal ions (1 mM) on SapHM activity.

SapHM activity was significantly inhibited by EDTA, EGTA, and DTT, and almost completely inactivated by the serine protease inhibitor PMSF, indicating that SapHM was likely a serine-type protease. In contrast, the enzyme activity was increased by 23% in the presence of 1 M urea (Table 3). The enzyme activity was activated by DMF and DMSO, and was stable in the presence of methanol and acetone. SapHM was also highly stable in the presence of various surfactants (Table 4). The non-ionic surfactant Tween 80 enhanced the enzyme activity by up to 121%.

Table 3.Effects of organic solvents and surfactants on SapHM activity.

Table 4.Hydrolysis of various substrates by SapHM.

Furthermore, substrate specificity analysis showed that SapHM was active on casein, BSA, and BSF. The highest activity was exhibited on casein, with moderate activity on BSA and BSF at 51% and 31%, respectively (Table 4). The Km and Vmax of SapHM toward casein at 75℃ were 35.7 mg/ml and 5 × 104 U/mg, respectively. No enzyme activity was observed when gelatin was used as a substrate.

Zymogram and Sequence Analysis

Zymogram analysis revealed a clear transparent halo on casein agar plates (lane 2), and the corresponding strip was SapHM on lane 1, which was stained with Coomassie Brilliant Blue (Fig. 3B). The target strip on lane 1 was excised for peptide mass fingerprinting. The peptide mass fingerprint was used as a query against the NCBI protein database (MASCOT search), and the results are represented in Table 5. There were two peptide fragments of SapHM by peptide mass fingerprint analysis: NH2-VSEAITVGATQSNDSR and NH2-ASYSNWGA TVDIFAPGTSITAAWR, which exhibited 100% identity with a hypothetical protein (WP_019431870.1) from Streptomyces sp. AA0539. In addition, the sequence was identically matched to peptidase S8 from Streptomyces sp. NRRL F-2890, alkaline serine protease from Frankia alni ACN14a, and peptidase S8 from Frankia sp. QA3 (Table 5). Furthermore, the gene encoding the protease SapHM can be obtained through the results of peptide fingerprinting, as described below.

Table 5.Results of peptide mass fingerprint analysis.

From the analysis of the peptide mass fingerprint, SapHM exhibited 100% identity with a hypothetical protein (WP_019431870.1) from Streptomyces sp. AA0539. Thus, specific primers were designed to amplify the protease gene of Streptomyces sp. M30 based on the gene sequence of the hypothetical protein from Streptomyces sp. AA0539. After the amplified PCR fragment was sequenced, the gene sapHM was 1,179 bp, corresponding to 392 amino acids with a predicted molecular mass of the full-length sequence as 39.4 kDa by BioEdit software. The first 24 amino acid residues were predicted to be a signal peptide, and the cleavage site was located between 24 and 25 amino acid residues. The gene of the mature peptide was 1,104 bp, corresponding to 368 amino acids with a predicted molecular mass as 37.1 kDa. The molecular mass by mass spectrometry was consistent with the theoretical molecular mass (Fig. 4). The nucleotide sequences of SapHM was deposited in the GenBank database.

The phylogenetic tree of alkaline protease SapHM is shown in Fig. 6. Similar proteins were aqualysin I from Thermus aquaticus Yt-1 (63% identity) and protein kinase K from Tritirachiurn album Limber (38% identity). Low identity exsited between them. Sequence alignment showed that the catalytic triad Ser-His-Asp, characteristic of serine proteases, was conserved in SapHM as well (Asp156, His187, and Ser339).

Fig. 6.A phylogenetic tree, generated by the neighbor-joining method, based on the SapHM amino acid sequences.

 

Discussion

Most alkaline proteases that have been reported are from high-yielding strains of Bacillus species [1,22]. In the study, SapHM, an extracellular alkaline protease from Streptomyces sp. M30, was purified and characterized by three major steps. The purification process is similar to that of protease from Geobacillus stearothermophilus [16] and catalase from Pigmentiphaga sp. DL-8 [10]. During the purification process, the main fractions with high activity were pooled, while other fractions with no or low activity were discarded to obtain the high-purity protease and simplify the purification procedure. This method will lead to low-yield and -fold purification, but the obtained sample is sufficient for further analysis and the yield (15.5%) was higher than the protease from Periplaneta americana (4.2%) [32]. The specific activity of purified SapHM was 29,070 U/mg, exhibiting higher protease activity than extracellular proteases from other Streptomyces, such as proteases from Streptomyces megasporus strain SDP4 (95.4 U/mg), Streptomyces sp. MAB18 (2,398.36 U/mg), and Nocardiopsis sp. (1,260 U/mg) [24,25,29]. The amino acid sequence of SapHM shared high identity with a hypothetical protein from Streptomyces sp. AA0539, and low identity with other reported proteases such as aqualysin I from Thermus aquaticus Yt-1 (63% identity) and protein kinase K from Tritirachiurn album Limber (38% identity). SapHM also contained a conserved catalytic motif (Ser-His-Asp), which also existed in other serine proteases. Thus, SapHM may be a novel protease, based on these special characterizations and low identity with other reported proteases.

In general, the molecular mass of alkaline proteases from microorganisms ranges 15-40 kDa. When SapHM was subjected to SDS-PAGE, the electrophoretic pattern of the protein molecular weight standard was altered. This phenomenon occurred repeatedly when the samples were heated at 90-100°C to fully inactive the protease. Considering the activity of SapHM was inhibited, this phenomenon could be caused by an unidentified substance that was introduced during the purification process. The amino acid sequences of mature peptide were predicted with the SignalP server and the molecular mass of SapHM was approximately 37.1 kDa. The molecular mass of SapHM was consistent with the result of the mass spectrometry (Fig. 4). Some alkaline proteases have been purified from microorganisms that had a similar molecular mass to SapHM; for example, protease from Bacillus circularns MTCC 7942 (43 kDa) [30], Aspergillus parasiticus (36 kDa) [3], and Thermus aquaticus YT-1 (38 kDa) [27].

SapHM showed activity at a broad temperature range (50-90°C) with an optimum temperature of 80°C (Fig. 5A). The optimum temperature of SapHM was significantly higher than actinomyces proteasetes, including S. corchorusii (70°C) [12], S. megaspores (65°C) [29], S. thermovulgaris (70°C) [38], and S. thermoviolaceus (50°C) [17]. It was also higher than proteases from other microorganisms, like Eap1 from Sporisorium reilianum (45°C) [23], alkaline protease from Stenotrophomonas maltophilia strain SK (40°C) [37], and protease from Aspergillus parasiticus (50°C) [3]. The thermostability of SapHM was advantageous, as substrate solubility was obviously increased and the risk of contamination was reduced significantly. These advantages are extremely beneficial for the detergent industry. Thus, this protease SapHM can be used as a detergent additive.

Most alkaline proteases from microorganisms are Ca2+ dependent, and they are inhibited by certain heavy metals [27]. Alkaline proteases from Bacillus brevis [4] and Thermoanaerobacter yonseiensis KB-1 [15] were strongly inhibited by Cu2+. In contrast, SapHM was insensitive to most heavy metals, and Cu2+ was the strongest activator of SapHM. Moreover, the protease activity was inhibited in the presence of chelating agents (EDTA and EGTA), indicating that SapHM was a metalloenzyme. SapHM was highly stable in the presence of Tween 80 and Triton X-100 (non-ionic surfactants), SDS and CTAB (anionic and cationic detergents, respectively), and the denaturant urea. In general, proteases are less stable in the presence of the strong anionic detergent SDS [2,35]; however, SapHM exhibited a slight increase in activity. SapHM also remained stable in the presence of the water-immiscible solvents DMF and DMSO. These characteristics of SapHM also showed that it could potentially be used in the detergent industry and leather industry.

In conclusion, we have isolated the protease-producing bacterium Streptomyces sp. M30, and characterized a novel thermostable alkaline protease. The alkaline protease SapHM may be used in various industrial applications, and further research regarding expression of the sapHM gene should be conducted.