|Peplist||Included in the Peplist with identifier PL00148|
|NC-IUBMB||Not yet included in IUBMB recommendations.|
|Preparation||First reports on isolation of stearolysin described the use of culture supernatants of G. stearothermophilius, adsorption onto Amberlite XAD-7, acetone precipitation and affinity chromatography (Eijsink et al., 1990) or ammonium sulfate precipitation and DEAE-Sephadex chromatography (Fujii et al., 1983). Higher yields were obtained by expression of the stearolysin gene in protease-deficient B. subtilis strains like MT-2 (deficient in the host's neutral protease gene (Fujii et al., 1983), DB104 (deletion of aprA3, inactivation of nprR2 and nprE18 genes by two point mutations (Kawamura & Doi, 1984) and DB117 (derivative of DB104, deletion of nprR2 and nprE18 genes, Eijsink et al., 1990). An efficient one-step isolation method of stearolysin and other metalloendopeptidases represents the use of columns with silica-coupled bacitracin (Van Den Burg et al., 1989). Another affinity chromatographic procedure applies the potent competitive inhibitor Gly-D-Phe bound to cyanogen-bromide activated Sepharose (O'Donohue et al., 1994, Eijsink et al., 1991). Alternative procedures use ammonium sulfate precipitation, DEAE-Sephacel chromatography and gelfiltration on Superdex75 HiLoad columns (Mansfeld et al., 1997). Another possibility, especially for the production of inactive variants, is the expression in Escherichia coli (Mansfeld et al., 2005). Renaturation from inclusion bodies after their solubilization by 6 M guanidine hydrochloride was unexpectedly possible in high yields without the folding assistance of the propeptide (Mansfeld et al., 2005).|
|Biotechnology||Fields of application of neutral proteases such as stearolysin are the baking (e.g. as gluten relaxer) and brewing industry, animal feed, food and beverage processing, production of dietary supplements and flavors, leather processing, enzymatic peptide synthesis (e.g. production of the aspartame precursor N-carbobenzoxy L-Asp-L-Phe methyl ester (Z-Asp-OMe) (Kuhn et al., 2002, Murakami et al., 2000) any other processes in down-breaking of proteinaceous raw materials (e.g. for restoration of artwork) at neutral pH.|
|Specificity||Stearolysin has a relatively broad substrate specificity comparable to all thermolysin-like proteases. Used substrates for activity assays: mostly proteinaceous substrates, such as casein (Fujii et al., 1983), hide powder Azure (Sidler & Zuber, 1972), Azocoll (Van Den Burg et al., 1989), an azo dye-impregnated collagen or more specific peptide substrates such as Z-Gly-Leu (Matsubara, 1966), furylacryloyl-modified peptides (especially 3-(2-furyl)acryloyl-Gly-Leu-amide, FAGLA (Feder, 1968, Mansfeld et al., 1997) or the intramolecularly quenched 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide (Abz-AGLA-Nba) (Nishino & Powers, 1980, Durrschmidt et al., 2010). Activity assay with FAGLA and other FA-modified di- and tripeptides is based on the spectrophotometric measurement of the decrease in absorbance at 345 nm due to cleavage of the Gly-Leu or Gly-Phe peptide bond with limitations in the determination of kinetic parameters for FAGLA due to the relatively high Km value (30 mM for thermolysin) in relation to its poor solubility (2 mM in 10% DMSO). Tripeptides with higher solubility can alterantively be used (de Kreij et al., 2001). With Abz-AGLA-Nba as substrate the increase in fluorescence emission at 415 nm (excitation at 340 nm) due to hydrolysis of the Gly-Leu bond is continuously measured. Stearolysin preferentially cleaves peptides and proteins at the N-terminal side of large hydrophobic amino acid residues preferably Phe at P1' position. Substrate specificity of all thermolysin-like proteases is predominantly defined by the hydrophobic S1" binding pocket (mainly Phe130, Phe133, Val139, Leu202 (de Kreij et al., 2000, de Kreij et al., 2001). The exchange of Phe133 by Leu as present in thermolysin shifted the substrate preference of stearolysin to that of thermolysin in terms of the ratio of kcat/Km values (de Kreij et al., 2001). Activity toward casein was increased twofold (de Kreij et al., 2000). The overall activity of stearolysin toward identical peptide substrates is generally lower than that of thermolysin (de Kreij et al., 2001). Possible reasons are differences in hinge-bending motions (Veltman et al., 1998) or changed active-site electrostatics. Enlargement of the binding pocket by replacement of Leu202 by smaller amino acids (Ala, Gly, Val) increased activity toward substrates with Phe at P1' but Leu202 is still the optimal residue for substrates with Leu at P1' position (de Kreij et al., 2001). Substrate specificity of stearolysin in enzymatic peptide synthesis is comparable to that of hydrolysis. The substrate preference at P1" position when using Z-Asp-OH (carboxyl component) in dipeptide synthesis and different amino acid According to peptide hydrolysis, thermolysin preferred Ile-OMe over Phe-OMe in peptide synthesis too because of the differences in the S1" subsite (Kuhn et al., 2002).|
The importance of the other subsites (S2, S1 and S2") is not known for stearolysin. With thermolysin, they are of minor importance (Hangauer et al., 1984, Morihara & Tsuzuki, 1970).
Neutral salts such as NaCl activate stearolysin as well as thermolysin in hydrolysis and synthesis (Kuhn et al., 2002, Inouye, 1992). An activating effect was also described for the cationic detergent cetyl trimethyl ammonium bromide (Mansfeld & Ulbrich-Hofmann, 2007). The modification of charged residues on the surface far away from the active site of the protein accelerated hydrolysis of peptides (de Kreij et al., 2002)up to 4-fold demonstrating the importance of long-range electrostatic interactions.
|pH optimum||Stearolysin has its maximum activity at neutral pH (Fujii et al., 1983, Sidler & Zuber, 1972).|
|Special substrate|| |
|Substrate comments||Proteinaceous substrates include casein (Fujii et al., 1983), hide powder Azure (Sidler & Zuber, 1972) and Azocoll (Van Den Burg et al., 1989). Specific peptide substrates include Z-Gly-Leu, (Matsubara, 1966), furylacryloyl-modified peptides (especially 3-(2-Furyl)acryloyl-Gly-Leu-amide, FAGLA) (Feder, 1968, Mansfeld et al., 1997) and 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide (Abz-AGLA-Nba) (Nishino & Powers, 1980, Durrschmidt et al., 2010)|
|Inhibitor comments||EDTA inhibits stearolysin irreversibly (Fujii et al., 1983, Sidler & Zuber, 1972) by release of the catalytically essential Zn2+ and the stability-determining Ca2+ ions. This leads to fast autoproteolytic degradation of the enzyme. Reversible inhibitors are zinc-chelating reagents such as 1,10 phenanthroline (mM concentrations) (Holmquist & Vallee, 1974) and various more specific inhibitors derived from di- or tripeptides with strong zinc-binding moieties such as phosphoramidon (J13.401). They bind specifically to Phe114 in the S1" subsite of thermolysin (de Kreij et al., 2000, Veltman et al., 1998). Even though not described for stearolysin, it probably will also be inhibited by the other highly specific inhibitors designed for thermolysin containing phosphonamidate, phosphoramidate, sulfhydryl, carboxylate, and hydroxamate groups. Stearolysin is also inhibited by aliphatic alcohols (methanol, ethanol, propan-1-ol, propan-2-ol) in hydrolytic as well as synthetic reactions (Kuhn et al., 2002). Propan-2-ol is used to prevent autoproteolysis in storage of these enzymes (Van Den Burg et al., 1989). The inhibitory effect of alcohols was shown to be caused by binding to the main subsites in the active site of the enzyme as demonstrated for thermolysin by soaking of crystals in 2–100% propan-2-ol (English et al., 1999). Furthermore stearolysin is competitively inhibited by guanidine hydrochloride (Ki 1 M at pH 7.5, 25°C) (Durrschmidt et al., 2001).|
|Structure||No three-dimensional structure is available. However, a homology model based on the three-dimensional structure of thermolysin (Matthews et al., 1972) was deposited in PMDB (id. PM0079201) (Castrignanò et al., 2006). Stearolysin consists of two domains - an N-terminal, mainly beta-stranded one and a predominantly alpha-helical C-terminal domain with the active site cleft between the two domains and the catalytically essential Zn2+ ion at the bottom of this cleft - as known for the other members of this family. The tetrahedrally coordinated Zn2+ ion (by His142, His146, Glu166) and an activated water molecule as nucleophile are important for catalysis. The central alpha-helix between amino acids 137-150 with the highly conserved His-Glu-Xaa-Xaa-His (HEXXH) motif connects the two domains. Glu166 determining stearolysin to be a glu-zincin, is located in an alpha-helix of the C-terminal domain. Glu143 as the general base in the catalytic mechanism is directly involved in catalysis (Hangauer et al., 1984). The conserved Gly 78, 135, 136 residues ensure the flexibility of the active site (Veltman et al., 1998) and are important for catalytic activity. The four Ca2+ ions of stearolysin are important for thermal stability as already shown by Sidler and Zuber (Sidler & Zuber, 1972) Two of the Ca2+ ions (Ca1 and 2) are bound in a highly conserved double-binding site close to the catalytic Zn2+ ion in the active site and the other two Ca2+ ions in single calcium-binding sites (Ca3 and Ca4) which are absent in the thermolabile enzymes of this family. The occupancy of the double calcium-binding site is an absolute prerequisite for correct folding (Dahlquist et al., 1976). Ca3 is bound to a stability determining surface-located loop structure (amino acid residues 55-69) via Asp57 and Asp59 side chains and Gln61 backbone carbonyl group. Ca4 is bound to a surface-located loop in the C-terminal domain via Asp200 and Thr194 side chains and backbone carbonyl groups of residues 193, 194 and 197. Single exchanges of amino acids involved in calcium binding in Ca3 and Ca4 reduced thermal stability but thermal stability of the mutant proteins was still strongly dependent on Ca2+ concentration. This could be overcome by mutations in the Ca3 binding site alone (Veltman et al., 1998) and additional stabilizing mutations in the vicinity of this site could compensate the observed stability loss caused by mutations in the Ca3 binding site (Veltman et al., 1997). In general, a strong stabilization effect was observed for mutations which decreased the flexibility of the region around Ca3 and were able to prevent local unfolding processes. Extremely stable variants of stearolysin were created (Mansfeld et al., 1997, Van den Burg et al., 1998) e.g. by the replacement of the amino acid residues Ser65 and Ala69 by Pro and the introduction of a disulfide bridge connecting this loop with the N-terminus (Mansfeld et al., 1997). This disulfide bridge could mimic the stabilizing effect observed upon occupancy of Ca3 site at high Ca2+ concentrations (Durrschmidt et al., 2005) by preventing the formation of a locally unfolded intermediate state. The most stable enzyme variant Boilysin (Van den Burg et al., 1998) which retains considerable activity at 100°C (half-life of 170 min at this temperature) and exceeds thermal stability of thermolysin considerably was obtained by eight amino acid exchanges only.|
|Physiology||As with the other extracellular, neutral proteases of this family, stearolysin degrades extracellular proteins for nutritional purposes of the bacteria.|
|Biological aspects||Stearolysin is synthesized as a preproprotein (Takagi et al., 1985, Kubo & Imanaka, 1988). An FTP (Fungalysin/Thermolysin Propeptide motif and a PepSY (Peptidase propeptide and YPEB) domain have been identified in the propeptide of stearolysin. The signal peptide (25 amino acid residues) is released during the Sec-controlled secretion process (Takagi et al., 1985). The prosequence comprises 204 amino acids and is released intramolecularly and autocatalytically after completion of the folding process as demonstrated for thermolysin (Marie-Claire et al., 1998) The mature protein comprises 319 amino acids. In B. subtilis, maximum expression of stearolysin occurs in the late exponential and early stationary phase before start of sporulation (Priest, 1977).|
|Contributing authors||Johanna Mansfield, Martin-Luther University, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.|