2.1 Strains, culture medium and growth conditions
Haloarchaeobius sp. FL176 was isolated from a dried seafood (salted-kelp) sample collected from a local supermarket in (Wuhu, China), in 2019. Strain FL176 was cultured using JCM 168 halobacteria medium (https://jcm.brc.riken.jp/en/) which contained (per liter) casamino acids (BD-Difco) 5.0 g, yeast extract (BD-Difco) 5.0 g, sodium glutamate 1.0 g, trisodium citrate 3.0 g, MgSO4·7H2O 20.0 g, KCl 2.0 g, NaCl 200.0 g, FeCl2·4H2O 36.0 mg, and MnCl2·4H2O 0.36 mg. The pH was adjusted to 7.0-7.2. Escherichia coli strains JM109 and BL21 (DE3), and strains derived from these were routinely cultured in Luria-Bertani (LB) medium [21]. For preparation of solid media, 15.0 g/L agar (Aobox, China) was added. Autoclaving was performed at 121°C for 15 min. If necessary, ampicillin (final concentration: 100 μg/mL) was added. Strain FL176 and E. coli strains (Table 1) were cultivated at 37 °C with rotary shaking at 200 rpm (for liquid medium).
To check whether cells of strain FL176 would lyse or not in distilled water, 1 mL of a cell suspension in the exponential growth phase was used to cells harvest through centrifugation at 12,000 rpm for 3 min. Next, the cell pellets were suspended with 1 mL ddH2O, and then centrifugation at 12,000 rpm for 3 min. Photographs were taken for showing the cell lysis.
2.2 Extracellular protease activity detection
Skim milk agar plates were prepared using the following steps. A 10 mL volume of 10% skim milk (BD-Difco) solution was heated in a sterile test tube in boiling water for 15 min prior to being added to 100 mL of autoclaved JCM 168 medium (with agar) that had been cooled to ~55-60 °C. After mixing, the medium was gently poured into petri dishes and left to solidify at room temperature (25 °C). Gelatin agar plates contained JCM 168 solid medium with the addition of 10% gelatin, and were autoclaved at 121°C for 15 min. Plates were inoculated with strain FL176 and incubated for 10-day’s at 37°C. Skim milk plates were inspected visually for clearing around colonies while gelatin agar plates were first flooded with Frazier reagent and left for 1 minute to detect protease activity [22].
2.3 Genome sequencing
Genomic DNA was extracted by using the TIANamp Bacteria DNA Kit according to the manufacturer's instructions (TIANGEN, China). The genomic DNA The genomic DNA that meets the requirements for sequencing was sequenced by a commercial sequencing company (Biomarker Technologies, China) using Illumina HiSeq 4000 platform. The quality control of the reads was performed using the FastQC version 0.10.0. The quality-filtered sequence reads were assembled into contigs by Velvet [23]. The completeness of each genome was calculated using CheckM version 1.0.7 with default options [24]. The assembled genome was deposited in the GenBank after annotation with Prokka version 1.14.5 [25], used to determine the extracellular protease encoding gene.
2.4 Phylogenetic analyses
To determine the taxonomic position of extracellular producing strain FL176, pair of primers specific for haloarchaea, F8/R1462, was used to amplify the haloarchaeal 16S rRNA gene [26]. PCR products were purified with a DNA gel extraction kit (Axygen, USA), and inserted to pMD-18T (TaKaRa, Japan) for DNA sequencing after transformation of E. coli JM109 competent cells and colony PCR verification by using the universal M13 F/R primers. Then, the 16S rRNA gene (MW290930.1) sequence of strain FL176 was used as the query to search the public databases of the 16S ribosomal RNA sequences (Bacteria and Archaea) via nucleotide BLAST (Basic Local Alignment Search Tool) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
For constructing the genetic relationship of strain FL176 within the genus Haloarchaeobius, the 16S rRNA gene sequences of other members in this genus and an out-group (16S rRNA gene of Halobacterium salinarum JCM 8978) were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/). Multiple sequences alignment was conducted using ClustalW embedded in BioEdit [27]. The phylogenetic relationship was inferred by MEGA7 [28] using the Neighbor-Joining method [29], bootstrap test (1000 replicates) [30], Kimura 2-parameter [31].
To show the active sites including the catalytic triad, functional motif and boundaries between different functional regions, multiple amino acid sequences alignments were performed among these coding gene known halolysins as well as the deduced halolysin(s) from Haloarchaeobius sp. FL176 using ClustalW embedded in BioEdit [27]. The data matrix of these aligned halolysins protein amino acid sequences was introduced to GeneDoc v.3.2 for sequence editing and visualizing. The data matrix was also used for construction the genetic relationship among Hly176A, Hly176B and other halolysins based on amino acid sequences using the same procedure as the 16S rRNA genes.
2.5 Cloning and protein expression of halolysin-like genes
The genome of Haloarchaeobius sp. FL176 was sequenced and deposited in GenBank (NCBI accession no. JANJYH000000000). To search for halolysin encoding gene(s), the genome was searched (BLASTn, https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the sequence of hlyR4 (D64073.1), the encoding gene of serine protease halolysin R4 (HlyR4) from Haloferax mediterranei ATCC 33500. Two putative genes (hly176A and hly176B) were detected that showed 70-71% nt sequence identity with hly176A. These were located on contig NODE_2_length_556356_cov_21.8158_ID_223 (498,162-499,014 bp and 498,162-499,014 bp, respectively). Primer pairs 176AF/176AR and 176BF/176BR were designed to amplify the entire hly176A and hly176B including the signal peptide region, respectively (Table 2). PCR amplification was performed using KOD DNA polymerase (TOYOBO, Japan) for 35 cycles consisting of 94 °C for 0.5 min, 53 °C for 0.5 min, and 72 °C for 1.5 min, followed by a final extension step of 7 min at 72 °C. The PCR products were purified and then digested with NdeI and EcoRI (Thermo, Lithuania). The digested fragments were inserted into the pET-22b(+) vector (Novagen, USA) with NdeI and EcoRI sites resulting in plasmids pET-22b-176A and pET-22b-176B (Table 2), and then transformed into E. coli BL21 (DE3) for protein expression.
Directed mutations of single codons of Hly176B were as follows. D160A, H199Q, and S352T targeted residues of the catalytic triad (Asp160-His199-Ser352). C286G altered the cysteine at position 286 to a glycine. These mutations were constructed by overlap extension PCR [21] using primer pairs D160AF/D160AR, H199QF/H199QR, S352TF/S352TR, and C286GF/C286GR combined with 176BF and 176BR, respectively. Deletion mutants were constructed using the same method the point mutations except that specific primer pairs were used. The deletion mutant Δ80 (80 aa deletion from 403 to 482 aa) used primer pair Δ80F and Δ80R. The signal sequence deletion mutant ΔS of Hly176B used primers 176BΔSF and 176BR. The mutant ΔCTE (a C-terminal extension (CTE) domain deletion of Hly176B) used primers 176BF and Δ176BCTE.
The mutated genes were inserted to the pET-22b (+) expression vector, verified by DNA sequencing, and introduced into E.coli BL21 (DE3). The recombinant strains of E. coli were cultivated in 100 mL of LB medium containing 100 μg/mL ampicillin at 37 °C, with shaking (200 rpm). When the OD600 reached 0.6-0.8, IPTG was added to 1 mM and the cultures incubated 30 °C for 5 h, after which the cells were harvested by centrifugation at 4 °C for 10 min at 12,000 rpm.
2.6 Protein purification from E. coli cells
To purify the recombinant proteins, the centrifuged cell pellets were resuspended in lysis buffer (8M Urea, 50 mM Tris-HCl, 10 mM CaCl2, pH 8.0) and disrupted by sonication at 0 °C for 20 min (ultrasonic processing 5 s followed by a pause of 5 s) using a SM-1000D sonicator (Shunma Tech, China), after which the cell debris was removed by centrifugation (10,000 rpm, 10 min, 4 °C). The cell-free extract was filtered through a 0.22 μm hydrophilic pore size nylon membrane filter (Millipore, USA), and the filtrate was then loaded onto a Ni-affinity chromatography column that had previously been chelated with Ni2+ and equilibrated with the lysis buffer. Unbound proteins were eluted from the column with washing buffer (8M Urea, 50 mM Tris-HCl, 10 mM CaCl2, 20 mM imidazole, pH 8.0). Then, the bound proteins were eluted from the column with an elution buffer (8M Urea, 50 mM Tris-HCl, 10 mM CaCl2, 100 mM imidazole, pH 8.0) [32]. The quality and purity of affinity-purified proteins were assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)[21].
2.7 Western blotting
To determine authenticity of the expression proteins, protein bands on SDS-PAGE were transferred to a nitrocellulose membrane and subjected to immunoblotting analysis using an HRP-conjugated anti-His mouse monoclonal antibody (Sangon Biotech, China) [21]. The target bands were shown using BeyoECL Moon chemiluminescence detection kit (Beyotime, China) according to the product’s operation instruction.
Qualitative or semi-quantitative detection of the protease activity was performed on skim-milk agar plate. To determine the proteolytic activity of recombinant proteins, 200 μL of each purified, refolded protein (~1 mg/mL) was loaded into pre-perforated holes (6 mm in diameter) in a skim-milk agar plate. Plates were incubated at 37 °C for 3 days to allow zones of clearing around the holes to develop.
Quantitative detection of the protease activity was conducted according to the azocaseinolytic assay [33], which was described by Stepanov et al. [33] and modified by Du et al. [32]. The reaction was incubated at 37 °C for 60 min in 400 μL of reaction mixture containing 0.5% (w/v) azocasein (Sigma) and 0.2 μg of enzyme in high salt buffer (2 M NaCl, 100 mM Tris-HCl, pH 8.0). The reaction was stopped by the addition of 400 μL of 10% (w/v) trichloroacetic acid (TCA). After incubation at room temperature for 30 min, the mixture was centrifuged to remove the precipitate. The absorbance of the supernatant was then measured at 335 nm (A335) with a UV-5100 spectrophotometer (Metash, China). As controls, azocasein and enzyme were incubated separately. One unit (U) of azocaseinolytic activity was defined as the amount of enzyme required to increase the A335 by 0.01 per minute. Protein concentration was determined by using Coomassie Plus (Bradford) Assay Kit (Thermo Fisher).
2.8 Effect of PMSF and DTT on halolysin activity
The urea in the protein solution after affinity chromatography was removed via dialysis. Samples of denatured protein were then diluted 10-fold with renaturation buffer containing 4M NaCl, 50 mM Tris-HCl, 10 mM CaCl2 and incubated at 37 °C for 60 min to allow the protein refolding [32].Proteolytic activity of the heterogeneously expressed Hly176B was determined using the azocaseinolytic activity assay [32] in the presence of 0, 0.01, 0.1, 1 and 10 mM PMSF (phenylmethylsulfonyl fluoride) or DTT (dithiothreitol) with an additional concentration of 0.001 mM. The NaCl concentration and pH in the purified Hly176B solution were 4 M and 8.0, respectively. Three parallel assays were performed.
2.9 Effect of NaCl, temperature and pH on enzyme activity
The optimum NaCl concentration for Hly176B proteolytic activity was determined using the azocaseinolytic assay with NaCl concentrations ranging from 0.3 to 4.0 mol/L (0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mol/L). The temperature and pH were adjusted to 8.0 with Tris-HCl (50 mM, pH 8.0) and 25 °C (room temperature), respectively.
To probe the effect of increasing temperature on the stability of Hly176B, the purified proteins were incubated at various temperatures (4 °C, 15 °C, or 30–75 °C at 5 °C intervals) for 10 min prior to determining enzyme activity using the azocaseinolytic assay. The NaCl concentration and pH in Hly176B solution was 4 mol/L and pH 8.0, respectively.
To obtain a pH range from 5.0 to 11.0 (at intervals of 1.0) of Hly176B solution, the pH of enzyme solution after refolding was adjusted to 6.0 with phosphate sodium hydroxide buffer (0.05M), and 7.0, 8.0, and 9.0 with Tris-HCl buffer (0.05M), and 10.0 and11.0 with borax sodium hydroxide buffer (0.05M). Protease hydrolysis reactions were carried out at the optimum temperature and NaCl concentration.
Azocaseinolytic assay was performed after 10 min under above conditions. Three parallel assays were performed to determine these optimum conditions of NaCl concentration, temperature and pH for Hly176B executing its highest proteolytic activity.
2.10 Effect of metal ions, detergents, chelating and other agents on enzyme activity
To test the influence of metal ions on the proteolytic activity of Hly176B, the concentration of ions was adjusted to 5 mM with a stock solutions (1 mol/L) of Ca2+ (CaCl2), Mn2+ (MnCl2), Cu2+ (CuSO4), K+ (KCl), Mg2+ (MgSO4), Fe2+ (Fe2(SO4)3), Zn2+ (ZnSO4) and Ni2+(NiSO4), respectively. Three detergents (Tween-20, Tween-80 and Sodium dodecyl sulfate (SDS), two divalent cation chelating agents (ethylene diamine tetraacetic acid, EDTA; and ethylene glycol tetraacetic acid, EGTA) and a serine protease inhibitor (phenylmethanesulfonyl fluoride, PMSF) were used. 50 mmol/L of EDTA, EGTA and PMSF, and 10% (w/v) of SDS were prepared as the stock solutions. Then, added 10 μL of these organic regents’ stock solutions for every 90 μL of Hly176B solution. After 10 min incubation (25 °C), enzyme activity was determined by the azocaseinolytic assay, with three biological replicates.
2.11 Stability of Hly176B under different NaCl concentration, temperature and pH
To probe the stability of Hly176B, the azocaseinolytic assay was conducted after protein solution being treated with various conditions for 0, 30 and 60 min. Six different NaCl concentrations (0, 0.5, 1.0, 2.0, 3.0 and 4.0 mol/L) were adopted, and two temperatures (30 and 50 °C) and two pH values (pH 6.0 and 8.0) were used. Three biologicals replicates were used.
To test the proteolytic activity of the crude enzyme, after cultivation in JCM 168 medium for 1 week with normal condition (see 2.1 Strains, culture medium and growth conditions), the culture supernatants (2L) of Haloarchaeobius sp. FL176 was collected after a centrifugation at 12,000 r/m for 3 min. Then, the supernatants was concentrated to 20-fold by using tangential flow filtration (TFF) with a molecular weight cut -off of 10 kDa. The proteolytic activity of concentrated supernatants (100 mL) was measured as the initial value. Then, the 20-fold concentrated supernatants was divided into two parts with equal volume, and stored in 4 °C and -20 °C respectively. The proteolytic activity of the crude enzyme was detected using azocaseinolytic assay with an interval of 7 days. Three biological replicates were used.