Identification and characterization of a bacterial glutamic peptidase
© Jensen et al; licensee BioMed Central Ltd. 2010
Received: 27 May 2010
Accepted: 1 December 2010
Published: 1 December 2010
Glutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Previously, only glutamic peptidases derived from filamentous fungi have been characterized.
We report the first characterization of a bacterial glutamic peptidase (pepG1), derived from the thermoacidophilic bacteria Alicyclobacillus sp. DSM 15716. The amino acid sequence identity between pepG1 and known fungal glutamic peptidases is only 24-30% but homology modeling, the presence of the glutamate/glutamine catalytic dyad and a number of highly conserved motifs strongly support the inclusion of pepG1 as a glutamic peptidase. Phylogenetic analysis places pepG1 and other putative bacterial and archaeal glutamic peptidases in a cluster separate from the fungal glutamic peptidases, indicating a divergent and independent evolution of bacterial and fungal glutamic peptidases. Purification of pepG1, heterologously expressed in Bacillus subtilis, was performed using hydrophobic interaction chromatography and ion exchange chromatography. The purified peptidase was characterized with respect to its physical properties. Temperature and pH optimums were found to be 60°C and pH 3-4, in agreement with the values observed for the fungal members of family G1. In addition, pepG1 was found to be pepstatin-insensitive, a characteristic signature of glutamic peptidases.
Based on the obtained results, we suggest that pepG1 can be added to the MEROPS family G1 as the first characterized bacterial member.
Biotech industries are becoming more and more successful in providing enzymatic solutions to an ever increasing number of industrial processes. The combination of high-throughput screening methods and the low cost of full genome sequencing has greatly sped up the process of identifying and isolating genes that match the criteria for a given industrial process. Besides being able to catalyze the enzymatic reaction in the industrial process, the enzymes must also be able to survive the often harsh industrial conditions. One of the frequently required capabilities of an industrial enzyme is the ability to function at high temperatures in either an acidic or alkaline environment. Enzymes with such properties can either be designed in silico or by high-throughput screening of microorganisms. High-throughput screening is often the first choice because optimization of an existing enzyme to an industrial process is much simpler than in silico design. The high-throughput screening is performed at conditions made to mimic the industrial process in order to find existing enzymes already able to cope with the industrial environment. Again, these study enzymes are often found in microorganisms that are able to grow in extreme conditions. By taking advantage of the many published and freely available genomes, it is often possible to make an educated guess of which microorganisms would be interesting to screen for a certain enzyme. Screening of such microorganisms will often provide an extensive battery of enzymes optimized for the selected screening conditions.
A soil screening conducted by Novozymes A/S resulted in the discovery of a novel strain of Alicyclobacillus (WO 2005/066339). The thermoacidophilic bacterial strain was isolated at low pH (approx. 4.5) and high temperature (60°C). The genus was identified by 16 S rRNA analysis and showed a significant phylogenetic distance from the previously known strains of Alicyclobacillus (WO 2005/066339). The strain was deposited in the DMSZ bacteria collection as Alicyclobacillus sp. DSM 15716. A gene for a putative G1 peptidase was identified in a gene library screening for secreted enzymes using Transposon Assisted Signal Trapping (TAST)  of Alicyclobacillus sp. DSM 15716 (WO 2005/066339).
The peptidase showed significant sequence similarity to the peptidase family G1 , a family otherwise thought to be limited to the filamentous fungal species of the Ascomycota phylum . The characterized proteins known to be part of the G1 family are aspergilloglutamic peptidase (AGP) from Aspergillus niger , scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum , acid peptidases B and C (EapB and EapC) from Cryphonectria parisitica , Penicillium marneffei acid proteinase (PMAP-1) , Talaromyces emersonii glutamic peptidase 1 (TGP1)  and BcACP1 from Botryotinia fuckeliana .
Based on sequence homology, five bacterial and a single archaeal protein have been annotated as putative G1 peptidases at the MEROPS peptidase database, but biochemical characterizations have not been carried out to confirm their function . Structural homology to fungal G1 peptidases and conservation of the catalytic residues indicate that pepG1 from Alicyclobacillus sp. DSM 15716 could be a bacterial G1 peptidase. In order to further examine its properties, we have amplified pepG1 from Alicyclobacillus sp. DSM 15716 genomic DNA and heterologously expressed it in B. subtilis. Following purification, pepG1 was characterized according to its physical properties, such as pH and temperature optimum and the effects of various protease inhibitors were determined. Based on these results, we suggest that pepG1 can be annotated as a G1 peptidase.
Results and discussion
Phylogenetic analysis of peptidase family G1
Catalytic residues and secondary structure of pepG1
Before the determination of the crystal structures of AGP and SGP, several attempts at elucidating the catalytic residues of G1 peptidases were carried out. Site-directed mutagenesis of conserved acidic residues was completed and the mutated enzymes were evaluated based on their catalytic activity. It is also known, that both AGP and SGP are expressed as precursor proteins which are autocatalytically processed into mature proteins in acidic conditions. By selecting both mutants unable to catalyze the conversion of precursor into mature protein, and those lacking peptidase activity, a glutamine (Q107 in SGP, Q133 in AGP) and a glutamate (E190 in SGP, E219 in AGP) were believed to be the active site residues of G1 peptidases [11–13]. The almost simultaneous publications of the near identical crystal structures of SGP and AGP verified the site-directed mutational studies and confirmed that the catalytic dyad in G1 peptidases consists of a glutamine and a glutamate residue [14, 15].
Protein signatures of known and hypothetical family G1 peptidases
Identification and expression of pepG1
The gene for a putative G1 peptidase was identified in a gene library screening for secreted enzymes using Transposon Assisted Signal Trapping  of Alicyclobacillus sp. DSM 15716 (WO 2005/066339). The gene encoding pepG1 was PCR amplified from genomic DNA of Alicyclobacillus sp. DSM 15716 and integrated by homologous recombination into the chromosome of B. subtilis MB1053. The signal peptide of pepG1 was replaced with a subtilisin-signal peptide for improved secretion in the B. subtilis host. SignalP cleavage site prediction for pepG1 was L33DA-SP . Expression of pepG1 was tested in three different liquid medias at two different temperatures. Fermentation was continued for up to six days. The highest peptidase activity at pH 3.4, 50°C towards AZCL-collagen was observed after five days of growth in PS-I media. Degradation of AZCL-Collagen resulted in the formation of a blue halo. The diameter of the halo was used as a rough measurement of activity.
Purification of pepG1
Characterization of pepG1
Effects of protease inhibitors and divalent cations on pepG1 activity
Class-specific inhibitors effect on pepG1 activity
pepG1 was incubated for 30 min with the below inhibitors at pH 4.0 (10 min, pH 4.5 for E-64). The remaining activity was assayed at 37°C.
Effect of divalent cations on pepG1 activity
pepG1 was incubated for 30 min with the below cations at pH 4.0. The remaining activity was assayed at 37°C, pH 4.0
Here we report the first characterization of a non-eukaryotic glutamic protease from the bacteria Alicyclobacillus sp. DSM 15716. Alignment of pepG1 with the known members of peptidase family G1 showed that the catalytic dyad, Q117 and E199 (pepG1 numbering) was conserved which indicates that the enzymatic mechanism is comparable to the fungal enzymes of this family. In addition, the crystal structure of SGP identified seven highly conserved motifs of the polypeptide chain clustered around the active and substrate-binding site of SGP . These motifs are highly conserved in pepG1. Furthermore, protein structure prediction of pepG1 by Phyre  found SGP and AGP to be the closest homologs, which was supported by homology modeling of pepG1. Very high structural similarities were observed between the homology model of pepG1 and the crystal structures of AGP and SGP [14, 15]. A number of protein signatures have been linked to G1 peptidases and three out of four are present in pepG1, despite the otherwise low sequence homology between pepG1 and the fungal G1 peptidases. The fourth signature could be identified by manual alignment and annotation of pepG1. The above bioinformatic studies of pepG1 clearly support the entry of pepG1 into the peptidase family G1.
To further validate the identity of pepG1, pepG1 was cloned into the expression host B. subtilis. Following expression and purification of pepG1, the pH and temperature optima of the peptidase and its stability were tested. In agreement with all G1 peptidases, pepG1 exhibited highest activity in acidic conditions. pepG1 was found to be resistant towards serine, cysteine, metallo and aspartic class-specific inhibitors, including pepstatin. Insensitivity to Pepstatin is a hallmark feature of all G1 peptidases.
Blast searches of the pepG1 sequence at NCBI identified several other putative bacterial G1 peptidases. If disregarding pepG1 homologs from related Alicyclobacillus species, new pepG1 homologs are found in the bacterias Amycolatopsis mediterranei, Geobacillus sp. and Catenulispora acidiphila along with archaeal homologs from Acidilobus saccharovorans and Picrophilus torridus. All of these homologs are between 40-50% identical to pepG1 and the active site residues, Q and E, that together form the catalytic dyad [14, 15], are conserved in all homologs. The in vivo function of G1 peptidases in bacteria and archaea is presently unknown. The majority of the fungal species secreting G1 peptidases are pathogens [6–9], in which the peptidases are most likely used to facilitate host tissue penetration and colonization by degrading structural proteins of the plant cell wall . The habitat of many of the microorganisms secreting G1 peptidases is soil or in some cases more extreme habitats, such as high temperature acidic environments. An obvious function could be scavenging as suggested by Fütterer et al, who sequenced and annotated the genome of the thermoacidophilic archaea, Picrophilus torridus .
The characterization of pepG1 presented in this manuscript along with the demonstrated presence of putative G1 peptidase homologs in an increasing number of non-fungal organisms strongly suggests that the non-fungal peptidase G1 homologs assigned to the MEROPS peptidase family G1 are correctly annotated.
All annotated and putative family G1 peptidases (except the non-peptidase homologues) in the MEROPS peptidase database (version 9.1)  as well as putative G1 peptidases identified by blast search at NCBI were aligned using ClustalX version 2.0.11. Bootstrapped maximum likelihood (100 iterations) phylogenetic tree was generated using ClustalX and PhyML 3.0 aLRT http://www.phylogeny.fr , respectively. Phylogenetic trees were visualized using TreeView http://taxonomy.zoology.gla.ac.uk/rod/treeview.html . Protein signatures in the bacterial and archaeal peptidases were identified using InterProScan  and ProDom . Sequence logo of the protein signature PR00977 was visualized using WebLogo version 2.8.2 . A model spanning residues 65-263 of pepG1 was generated using SWISS MODEL . The model structure was based on the PBD-file 2ifw and subsequently verified using PROCHECK [19, 20] and Ramachandran maps generated by PDBSum . PYMOL http://www.pymol.org was used for visualizing the model structure of pepG1.
Bacterial strain and culture conditions
Alicyclobacillus sp. DSM 15716 was grown on ATBA-1 agar pH 4.5 (400 ml of 0.625% Tryptone (Difco), 0.625% amylopectin (ICN) and 2.5% agar, granulated (Difco) mixed with 100 ml of 0.1% ammonium sulfate, 0.25% magnesium sulfate, 0.125% calcium chloride and 1.5% potassium dihydrogen phosphate) at 60°C overnight.
Cloning of pepG1 into Bacillus subtilis MB1053
The gene encoding pepG1 was amplified by PCR from genomic DNA of Alicyclobacillus sp. DSM 15716 and integrated by homologous recombination in B. subtilis MB1053 (amyE, apr, npr), in which the native subtilisin peptidase has been knocked out (WO03/0956658). Homologous recombination was done using an integration cassette consisting of two regions (with homology to the integration site on the B. subtilis genome) that together flanked pepG1 under control of a triple promoter. The triple promoter system consists of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIA promoter . The two flanking regions were amplified from a modified B. subtilis MB1053 strain in which the Spectinomycin gene has been replaced with a marker gene encoding Chloramphenicol and a gene encoding the subtilisin protease, SAVINASE™. The 5'-flanking region covers the yfmD gene to the SAVINASE™-signal-peptide (included) and introduces an overhang to pepG1. The 3'-flanking region located downstreams from the SAVINASE™gene covers Pel(end)-yflS-citS and introduces an overhang to the 3'-end of pepG1. The B. subtilis MB1053 cell strain was made competent according to Yasbin et al .
Nucleotide sequence analysis
The DNA sequences from both strands were determined with the BigDye Terminator v3.1 Cycle Sequencing Kit (Perkin Elmer) and Applied Biosystems 3730 XL DNA analyzer according to manufacturer's instructions.
Selection of constructs for purification
The construct was grown in three different liquid media, PS-1 (10% sucrose (Danisco), 4% Soymeal (Cargill), 1% Na2HPO4•12H2O, 0.5% CaCO3 and 0.01% Dowfax 63N10), Cal18 (4% Yeast extract (Difco), 0.13% MgSO4•7H2O, 5% Maltodextrin (Roquette), 2% Na2HPO4•12H2O, 0.67% Na2MoO4 Trace metal solution and 0.01% Dowfax 63N10) and SK-1 M (4% Sodium Caseinate (MD-Food), 20% Maltodextrin, 5% Soybean meal and 0.01% Dowfax 63N10), all supplemented with 6 mg/L chloramphenicol. Fermentations were performed on rotary shaking tables in 500 ml baffled Erlenmeyer flasks each containing 100 ml liquid media at 37°C and 30°C. Samples were taken at day 2, 3 and 4 from Cal18 media and day 4, 5 and 6 from PS-1 and SK-1 M and analyzed for activity. The activity was determined by a spot test of 20 μl supernatant in 1% agarose plates at pH 3.4 with 0.1% AZCL-Collagen. The plates were incubated at 50°C over-night and activity was visible as a blue halo around the spots.
Fermentation and purification of A. sp. pepG1
Fermentation of B. subtilis expression clone was performed on a rotary shaking table in 500 ml baffled Erlenmeyer flasks each containing 100 ml PS-1 media supplemented with 6 mg/L chloramphenicol. The clone was grown for five days at 37°C. Culture broth was centrifuged (20000 × g, 20 min) and the supernatant was filtered through a Seitz EKS filter plate. The EKS filtrate was adjusted to a pH of 4.0 with citric acid and heated to 70°C with continued stirring in a water bath. The solution was immediately placed on ice after the temperature reached 70°C. The precipitate was removed by a second filtration using a Seitz EKS filter plate. (NH4)2SO4 was added to a final concentration of 1.6 M and the pool was applied to a Butyl-Toyopearl 650 S column (bed volume 30 ml) equilibrated in 20 mM CH3COOH/NaOH, 1.6 M (NH4)2SO4, pH 4.5. After washing the column extensively with the equilibration buffer, protein elution was done with a linear gradient between the equilibration buffer and 20 mM CH3COOH/NaOH, pH 4.5 with 25% 2-propanol. Fractions from the column were analyzed for protease activity at pH 4.0, 37°C and fractions with activity were pooled. The pooled fractions were transferred to 20 mM CH3COOH/NaOH, pH 5.5 on a G25 sephadex column and applied to a Source 30Q column (bed volume of 40 ml) equilibrated in the same buffer. After washing the column thoroughly with the equilibration buffer, the protease was eluted with a linear NaCl gradient (0 to 0.5 M) in the same buffer. Fractions from the column were analyzed for protease activity (pH 4.0, 37°C). An additional elution with 1.0 M NaCl, 20 mM CH3COOH/NaOH, pH 5.5 was performed in order to release the remaining pepG1 from the column and fractions with activity were pooled. The slightly colored pool was treated with 1% (w/v) activated charcoal for 5 minutes and then passed through a 0.45 μm filter. The purity of the filtrate was analyzed by SDS-page and protein concentrations determined using Bradford protein assay.
Automated Edman degradation of purified pepG1 was accomplished with a Perkin-Elmer ABI 494HT sequencer with online microbore phenylthiohydantoin-amino acid detection.
Protease enzyme activity was assayed using Protazyme OL (crosslinked and dyed collagen from Megazyme). A Protazyme OL tablet was suspended in 2.0 ml 0.01% Triton X-100 by gentle stirring. 500 μl of the Protazyme suspension and 500 μl assay buffer (100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100 pH 4.0) were mixed in an Eppendorf tube and placed on ice. 20 μl protease sample was added and the assay initiated by transferring the Eppendorf tube to an Eppendorf thermomixer set at the assay temperature. The tube was incubated for 15 min on the Eppendorf thermomixer at its highest shaking rate (1400 rpm) and the reaction was stopped by transferring the tube back into the ice bath. The samples were then centrifuged in an icecold centrifuge for 3 min at 20,000 g and 200 μL supernatant was measured at OD650. A buffer blind without enzyme was included in the assay. OD650(Enzyme) - OD650(buffer blind) was used to express enzyme activity.
The above assay was used to determine the pH and temperature effect on activity, pH stability and temperature stability. pepG1 temperature stability was determined by incubating the enzyme at 50°C, 60°C and 70°C. Samples were taken after 10, 30 and 60 minutes of incubation, cooled on ice and assayed at 37°C, pH 4.0 in order to determine residual activity. pH stability was determined by diluting pepG1 5× in assay buffer pH 2-12 (total volume 100 μl) followed by incubation at 37°C for 2 hours. After incubation, 440 μl assay buffer pH 4.0 was added and assay was performed as described above. pH of the assay buffer was adjusted by addition of either NaOH or HCl.
Effect of divalent metal ions on A. sp. pepG1 activity
Purified A. sp. pepG1 protease (20 μl) was incubated for 30 min in 500 μl citric acid buffer pH 4.0 (33 mM citric acid/17 mM sodium citrate and 0.01% Triton X-100) containing 5 mM concentrations of divalent ions. These samples were then assayed for activity with Protazyme OL suspended in 500 μl of citric acid buffer pH 4.0 containing a 5 mM concentration of the divalent ion at 37°C for 15 min.
Inhibitor studies on A. sp. pepG1
Purified A. sp. pepG1 protease (20 μl) was incubated with the inhibitors, Pepstatin, EDTA and PMSF, for 30 min in 500 μl universal buffer pH 4.0. E-64 treatment of pepG1 was carried out for 10 min in 20 mM CH3COOH/NaOH, 1 mM CaCl2, pH 4.5. All samples were assayed for residual activity with Protazyme OL tablets at pH 4.0 (pH 4.5 for E-64), 37°C with the inhibitors present at the same concentrations as during the incubation.
Family G1 peptidase pepG1 from Alicyclobacillus sp. DSM 15716 [GenBank: HM011103].
We would like to thank Professor Birger Lindberg Møller for critical review of this manuscript, Björn Hamberger for helpful discussions and Emma O'Callahan, Camilla Knudsen and Pernille Sølvhøj Roelsgaard for proofreading. The Faculty of Life Sciences, University of Copenhagen is acknowledged for granting a PhD stipend to KJ.
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