2.1 Isolation and screening of microorganisms
Sampling was done at the hydrothermal vent site at Kueishantao (also called Turtle Island), an island in the East China Sea, part of Toucheng Township, Yilan County, Taiwan. Kueishantao is situated 9.1 km east of Kengfang Fishery Harbor3. We focused particularly on the isolation of bacteria from the vent crab Xenograpsus testudinatus at Kueishantao. All the bacterial strains isolated from this vent crab were screened for protease production using agar plates based on skim milk by measuring the zone of hydrolysis. Based on the highest proteolytic zone produced on skim milk agar plates, a bacterial strain was selected that was sequenced using the bacterial barcoding gene, 16 s RNA gene, applying the universal primers 27F and 1492R15. The sequence was edited by chromas 2.2 software and BlastN sequencing was performed followed by the construction of a phylogenetic tree using MEGA-X software16.
2.2 Amplification of the serine protease gene
Primers used for the polymerase chain reaction were forward primer 5′ – CGGGATCCCACRAATACTTCAAGYGCTGA-3′ and reverse: 5′ –CGGAATTCGCATTGACTCTACCRTTTTTCCA-3′17. The genomic DNA was isolated using a genomic DNA isolation kit according to the instructions of the company (NucleoSpin® Microbial DNA, MACHEREY-NAGEL, Dȕren, Germany). PCR was performed with 2 µL of DNA extracted as a template (50 ng), 2.5 µM of each primer, 10X PCR buffer and 2 U of Taq polymerase (Invitrogen), 0.5 mM dNTPs in a 25 µL reaction. Polymerase reaction (T100 Thermal cycler, Bio-Rad, Hercules, California, USA) with initial denaturation at 95 °C for 5 min, repeated 34 cycles of denaturation at 95 ℃ for 1 min, annealing at 58 ℃ for 1 min, extension at 72 ℃ for 1 min and a final extension at 72 ℃ for 10 min was done. The amplified gene was eluted by a Gel purification kit (Mini Plus Plasmid DNA extraction System, Viogene, Taipei, Taiwan). The gel eluted product was send out for sequencing, and bacterial identification was confirmed using BLAST at NCBI the PCR product was used for cloning.
2.3 Transformation in E. coli host cells
The PCR product was cloned in T and A cloning vectors. The ratio of vectors to insert was 1:3. The ligation reaction was set up according to the manufacturers protocol. In brief, 10 µL of reaction volume with 10x diluted ligation buffer, 1 µL T4 DNA ligase insert to vector ratio of 3:1, respectively, was added and kept at 4 ℃ overnight. The ligation was confirmed by agarose gel electrophoresis. Transformation was carried out in one shot of E. coli (ECOS™ 101 DH5α) competent cells according to the manufacturers protocol. Briefly, the cells were thawed and 2.5 µL of ligation mixture were mixed and vortexed. The mix was incubated on ice and a temperature shock at 42 ℃ was provided for 40 sec. The cells were then plated on prewarmed plates with LB agar (0.5 mM IPTG, 40 µg/mL ampicillin, and 40 µg X-gal) and incubated overnight. Positive white colonies were selected and confirmed by colony PCR and plasmid sequencing.
Digestion of plasmid vector T and cloning vector A was done by HIND-III restriction enzymes in the presence of NEB buffer. The reaction was set up with vectors having 1000 ng concentration, enzyme 5 U, and buffer and were incubated overnight at 37 ℃. Restriction was confirmed by agarose gel electrophoresis and HIND-III used for ligation in the expression vector. The expression vector pET-32b (+) was also digested using HINDIII restriction enzyme and confirmed by agarose gel electrophoresis. The cut vector pET32b + and Insert was ligated using T4 DNA ligase enzyme at a ratio of 1:3, respectively, and incubated overnight at 4 ℃. The transformation was performed in one shot ECOS BL21 (DE3) E. coli cells, following the manufacturer’s instructions. Briefly, 3.5 µL of the ligated product was mixed with competent cells (E. coli BL21 (DE3)) and kept on ice for 5 min right after a heat shock of 42 ℃ was provided for 40 sec and plated on pre-prepared warmed plates with amp x-gal and IPTG. Blue white screening was used to identify positive colonies, colony PCR, and were finally confirmed by the Sanger sequencing method [18]. The BLASTN database of NCBI was used for sequence similarity search. Homology alignment was done with the Clustal Omega program. By selecting the sequence with the highest similarity a phylogenetic tree was constructed using Mega-X software16.
2.4 Optimization of induction condition for the expression of SLSP-k in E. coli (DE3)
Transformed E. coli BL21 (DE3) cells were grown in 10 mL of LB medium with 50 µg/mL of ampicillin at 37 °C by shaking overnight. The primary culture was inoculated into four 0 mL tubes at a ratio of 1:10. To determine the optimum induction temperature, recombinant E. coli BL21 (DE3) were grown at 37 °C until the absorbance 0.6 was reached at OD600. Then IPTG (0.5 mM – 1 mM) was added. Incubation took place at 37 °C and 27 °C with 0.5 mM IPTG and 1 mM IPTG at each temperature for up to 10 h. One mL sample was taken every 1 h from T3 to T10 at 37 °C and 27 °C. The cell pellets were suspended in phosphate buffer and sonicated for 5 minutes with 20 sec pulses. The samples were centrifuged at 13000 rpm for 10 minutes and the supernatants were analyzed by SDS-PAGE.
2.5. Lysis buffer selection
Five different lysis buffers, listed in Table 1, were used to lyse the cell pellets. The supernatant was analysed by SDS PAGE. The buffer with highest yield of soluble recombinant protein was selected for further studies. From each buffer 2 mL were added to the cell pellet from a 5 mL IPTG-induced culture and sonicated for 5 min duration. The resulting lysed sample was centrifuged at 13,000 rpm for 10 min and the supernatant was then purified.
Table 1
Buffers and their composition used in the expression optimization of recombinant SLSP-k.
S. No.
|
Buffer
|
Final concentration
|
1
|
Tris-HCl (Merck), pH 7.5
|
20 mM
|
Dithiothreitol (DTT) (Sigma-Aldrich)
|
0.1 mM
|
Lysozyme (Sigma-Aldrich)
|
1 mg/m
|
2
|
Tris-HCl (Merck), pH 7.5
|
20 mM
|
NaCl
|
0.5 mM
|
Lysozyme (Sigma-Aldrich)
|
1 mg/mL
|
3
|
Phosphate buffer
|
20 mM
|
NaCl
|
0.5 mM
|
Urea
|
8M
|
Triton-X 100
|
1%
|
4
|
Phosphate buffer
|
20 mM
|
NaCl
|
0.5 mM
|
Triton-X 100
|
1%
|
5
|
Phosphate buffer
|
20 mM
|
Triton-X 100
|
0.5 mM
|
2.6. Purification of recombinant SLSP-k enzyme
The supernatant was filtered on a 0.22 µm filter and eluted by Ni Sepharose 6 fast flow) resin in PD10 columns to elute the binded his-tagged proteins. Binding buffer with 20 mM imidazole eluted the binded his-tagged proteins and unbound proteins were washed using the washing buffer. Elution buffer at two different concentrations, 200 mM and 500 mM, was added to elute the his-tagged proteins to check for highest soluble recombinant proteins.
2.7. Zymography and SDS-PAGE
The molecular weight of the soluble recombinant purified SLSP-k protein was studied by SDS-PAGE with stacking gel (4%) and resolving gel (12%). Zymography to check protease activity using 10 mg/mL gelatin was performed. The zymography gel electrophoresis was run at 100 volts and 4 ℃ (BIO-RAD, Hercules, California, USA). The gel was washed in Triton X-100 (2.5%) solution at 37 ℃ for 30 min at gentle shaking. The gel was kept overnight in the developing buffer (pH 7.5) comprising of Tris base, CaCl2, ZnCl2, NaCl, and Brij 35 at 37 ℃. Coomassie brilliant blue R-250 (0.1%) was used for 1 h each staining and de-staining (water: methanol: glacial acetic acid at ratios of 5:4:1) until clear bands visibly appeared, indicating protease activity on the gel.
2.8. Mass spectrometry analysis of the purified protein
The band of the SDS gel was excised and destained. Trypsin digestion was performed at 37 °C for 4 h (In-Gel Tryptic Digestion Kit, Thermo Fisher Scientific) in order to identify the peptide sequence by mass spectrometry (MS). Desalting of the tryptic digested peptides were performed on a C18 proteomic column (Mass Solution Ltd., Taipei, Taiwan). MS analysis of the resulting peptides applying nLC/Q-TOF (Micromass, Manchester, UK) was performed. The resulting MS data were used to search against entries in the NCBI database using the MASCOT search program (Matrixscience, London, UK). Additionally, peptides with acetylated lysines were predicted. The parameters searched for were: mass values: monoisotopic; fragment mass tolerance: ± 0.4 Da; protein mass: unrestricted; maximal missed cleavages: 1; peptide mass tolerance: ± 0.4 Da; variable modification: oxidation in methionine; acetylation in lysine: carbamidomethylation in cysteine.
2. 9. Bioinformatic Analysis
Protein sequence similarity and phylogenetic analysis was done applying the blastp program at NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi. The sequences were selected on the basis of similarity percentage identity. For multiple sequence alignment we used the Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/). The I-TASSER structure prediction program was used to predict structures which used COFACTOR and COACH tools. COFACTOR can retrieve ligand-binding sites, EC and GO, by comparing the already available structures. Meta-server COACH provides output by combining data from multiple functional annotations (from the COFACTOR, S-SITE, and TM-SITE)19 (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). To determine the signal peptide region SignalP server was used (http://www.cbs.dtu.dk/services/SignalIP/). A Phylogenetic tree was constructed by MEGA-X software. The confidence of the branching value was tested by bootstrapping 500 iterations. The final structures were retrieved from Discovery studio program for high quality images20.
2.10. FT-IR analysis of casein hydrolysates
The hydrolysis of casein by SLSP-k was measured by highly sensitive FT-IR techniques. Enzyme and casein was mixed at equal volumes at optimal conditions, i.e. at 50 ℃ and pH 10.0 kept for 30 min. The hydrolysed product was centrifuged at 10,000 rpm for 10 min at 4 ℃ and the supernatant was collected. The obtained supernatant was freeze-dried overnight. FT-IR spectroscopy was performed by mixing 225 mg dried KBr (10% w/w) with 25 mg freeze dried hydrolysate.
2.11. Biochemical characterization
2.11.1. Protease Activity Assay
Proteolytic activity was assayed with casein (0.6%) as a substrate. The reaction was carried out with 1 mL of enzyme and 1 mL of substrate at 37 ℃ for 30 min. The reaction was stopped by adding 1 mL of 10% TCA (trichloroacetic acid), incubated at room temperature for 15–20 min and centrifuged at 5000 rpm for 10 min. Spectrophotometric absorbance reading was taken after mixing 1.0 mL of supernatant was mixed with 650 µL of 0.5 M Na2CO3 and 500 µL of two times diluted Folin-Ciocalteu reagent. The absorbance reading was taken by a UV spectrophotometer after 30 min of incubation at 660 nm against the blank sample.
2.11.2 Determination of optimum protease conditions
Enzyme activity was observed at varying temperatures ranging from 40–100 ℃. For this purpose, 500 µL of the 0.6% (w/v) casein solution was mixed with 500 µL of enzyme solution followed by incubation for 1 h. Activity was studied according to a standard assay at each temperature. The relative activity was measured by keeping the highest activity as 100%. Thermal stability was determined with 3500 µL of the enzyme solution being kept in a water bath at a temperature ranging from 40 to 100 ℃ for 7 h. From the total mixture, a volume of 500 µL of enzyme was taken for reading after every 1 h. The relative activity (%) was calculated from the absorbance value.
SLSP-k activity was measured at varying pH values ranging from highly acidic to alkaline (2–12 pH). Since the protein was eluted and showed maximum solubility in phosphate buffer, the same buffer was used to predict the optimal pH for hydrolytic activity. Diluted enzyme solution in respective buffer (500 µL) was mixed with 0.6% casein solution in a total reaction volume of 3 mL followed by 1hour water bath at 50 °C incubation. The highest absorbance value was accepted as 100% and the relative activity from the absorbance (%) was predicted.
To study the effect of inhibitors PMSF, EDTA, and DTT was used. The final concentration of inhibitors used were 1 mM and 5 mM. In this study 500 µL of inhibitor solution was stirred with 500 µL of enzyme solution and incubated for 30 min. Then the standard protease activity assay was performed and residual activity was calculated.
To study the stability of surfactants, 1 mM and 5 mM of SDS, Tween-20, Triton-X 100 was used. The surfactant solution, 500 µL, was added to 500 µL of enzyme solution and incubated for 1 h and later a standard protease assay was performed as mentioned in Sect. 2.11.1. The residual activity of SLSP-k was calculated.
The stability of SLSP-k was analysed after treatment with solvents like DMSO, ethanol, ethyl acetate, methanol, 2-propanol, acetone, acetonitrile, and NaCl. In this treatment, 100 µL of organic solvent were added to 900 µL of enzyme solution, kept for 1 h at 50 °C. A sample without the treatment of any organic solvent was kept as a control. We calculated the residual activity (%) of the enzyme from the absorbance value.
To find the effect on SLSP-k activity with the treatment of metal ions, such as monovalent metal ions (Na+ and K+), divalent metal ions (Ca+ 2, Co+ 2, Cu+ 2, Cd+ 2, Mn+ 2, Pb+ 2, Hg+ 2, Ni+ 2), and trivalent Fe3+ were used. Metal solutions (500 µL) at concentrations of 1 mM, 5 mM, and 500 µL of enzyme solution were mixed followed by incubation for 1 h at 50 ℃. The relative hydrolytic activity was predicted from the absorbance.
The activity of the SLSP-k for kinetic studies to calculate the Vmax and Km with varying final concentrations of casein as substrate was performed from 2–20 mg/mL in phosphate buffer with pH 10 at 50 °C. The maximum velocity Vmax and the Michaelis–Menten constant Km was calculated from Lineweaver–Burk plots21.
To check for keratinase activity, chicken feathers and human hair were treated with 500 µL of SLSP-k in phosphate buffer and incubated at 50 ℃ for 48 h. The samples were dried completely to remove excess water at 60 ℃ and using SEM analysis by drying and fixing the samples on carbon tape, and sputtering them with gold22.
Since human hair was used in the study our ethical compliance statements are required and stated as following: a) All methods were carried out in accordance with relevant guidelines and regulations; b) We confirm that all experimental protocols were according the institutional regulations, namely that there is no formal permit or licence required for hair harvested from the first author’ GR hairbrush for the above experiment; c) No written informed consent was needed since the only subject (GR - see above) providing human hair from her hairbrush is over 18 years of age – since no parent and legal guardian was required – obtained the hair without pain from her hairbrush.