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At least 19 recordsLinked to original sources
PubMed1978

The determination of molecular weights of Streptomyces subtilisin inhibitor and the complex of Streptomyces subtilisin inhibitor and subtilisin BPN' by sedimentation equilibrium.

The molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mM phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0 degrees C. The molecular weight of SSI was found to be 23,000 over a wide concentration range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been calculated to be 11,500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23,000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5 X 10(-7)-5 X 10(-4) M. The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78,000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponent-polydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11,500) and one mol of the enzyme (molecular weight, 27,500) are tightly bound (Kd less than 1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.

Bacterial Proteins↗
PubMed1981

Thermal denaturation of streptomyces subtilisin inhibitor, subtilisin BPN', and the inhibitor-subtilisin complex.

The thermal unfolding of the microbial proteinase inhibitor Streptomyces subtilisin inhibitor (SSI) [Sato, S., & Murao, S. (1973) Agric, Biol. Chem. 37, 1067-1074), the bacterial proteinase subtilisin BPN' (EC 3.4.21.14), and the complex formed by these two proteins has been studied by differential scanning calorimetry (DSC). The thermal denaturation of SSI at pH 7.00 is fully reversible while those of subtilisin BPN' and its complex with SSI are not. The DSC data show that dimeric SSI remains dimeric as the temperature is raised until it unfolds and that it then dissociates during the unfolding process. The apparent specific heat of denatured SSI decreases rapidly with increasing temperature, a behavior not previously observed for proteins. The shape of the DSC curves observed with the enzyme-inhibitor complex suggests that the two components of the complex undergo their unfolding transitions more or less independently. The enthalpies of unfolding of mixtures of enzyme and inhibitor in various molar ratios indicate a substantially large enthalph of interaction than that deduced from fluorescence titrations (Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202).

Bacterial Proteins↗
PubMed1998

Reasoning enantioselectivity and kinetics of seleno-subtilisin from the subtilisin template.

The active-site serine (Ser221) of subtilisin Carlsberg(from Bacillus licheniformis) and subtilisin BPN' (fromBacillus amyloliquefaciens) was chemically converted into a selenocystein. Contrary to subtilisin's protease activity the semisynthetic seleno-subtilisin catalyzed the reduction of hydroperoxides. Enantioselectivity and kinetics of this reaction were studied by kinetic resolution of five racemic alkyl aryl hydroperoxides catalyzed by the seleno-subtilisin variants. Due to the identical tertiary structure of subtilisin and seleno-subtilisin, the enzymes have comparable substrate binding properties. Thus, a rational screening for suitable peroxidase substrates featuring structural characteristics of known subtilisin substrates was enabled. The enantioselective recognition of (S)-configured alkyl aryl hydroperoxides by seleno-subtilisin was comprehensible by subtilisin's preference for comparable (S)-alkyl aryl amines or alcohols. The analysis of chiral products by multidimensional gas chromatography revealed enantiomeric excesses up to 98%. Kinetics of seleno-subtilisin were rationalized on the basis of the established substrate-catalyst interactions of the subtilisin framework. The Carlsberg and BPN' peroxidase variants revealed typical differences in turnover numbers (kcat) and Michaelis-Menten affinity constants (Km) already known from subtilisin variants. Turnover numbers of seleno-subtilisin BPN' were lower and Km values were higher in comparison to Carlsberg variant. Substrate affinity of several substituted 1-arylethyl hydroperoxides to seleno-subtilisin was reasonable in comparison to corresponding aryl boronic acid inhibitors of subtilisin.

Bacillus↗
PubMed1991

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution.

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.

Bacterial Proteins↗
PubMed1998

Engineering the independent folding of the subtilisin BPN' pro-domain: correlation of pro-domain stability with the rate of subtilisin folding.

The 77-amino acid pro-domain greatly accelerates the in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In this complex the pro-domain has a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helixes. When isolated from subtilisin, however, the pro-domain is 97% unfolded even under optimal folding conditions. The instability of the isolated pro-domain suggests that there may be a thermodynamic linkage between the stability of the pro-domain and its ability to facilitate subtilisin folding. On the basis of the X-ray crystal structure of the pro-domain subtilisin complex, we have designed stabilizing mutations in three areas of the pro-domain: alpha-helix 23-32 (E32Q), beta-strands 35-51 (Q40L), and alpha-helix 53-61 (K57E). These amino acid positions were selected because they do not contact subtilisin in the complex and because they appear to be in regions of the structure which are not well packed in the wild type pro-domain. Since none of the mutations directly contact subtilisin, their effects on the folding of subtilisin are linked to whether or not they stabilize a conformation of the pro-domain which promotes subtilisin folding. By sequentially introducing the three stabilizing mutations, the equilibrium for folding the pro-domain was shifted from 97% unfolded to 65% folded. By measuring the ability of these mutants to fold subtilisin, we are able to establish a correlation between the stability of the pro-domain and its ability to accelerate subtilisin folding. As the pro-domain is stabilized, the folding reaction becomes faster and distinctly biphasic. A detailed mechanism was determined for the double mutant, Q40L-K57E, which is 50% folded: P + Su if (30 800 M-1 s-1, 0.04 s-1) PSI if (0.07 s-1, <0.005 s-1) PS. PSI is an intermediate complex which accumulates in the course of the reaction, and PS is the fully folded complex. The more stable the pro-domain, the faster the folding reaction up to the point at which the isomerization of the intermediate into the fully folded complex becomes the rate-limiting step in the folding process.

Cloning, Molecular↗
PubMed1990

Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro.

The formation of active subtilisin E from pro-subtilisin E requires the removal of the N-terminal pro-sequence of 77 residues. Pro-subtilisin E produced in Escherichia coli using a pINIII-ompA vector was first extracted with 6 M guanidine-HCl and 5 M urea and purified to homogeneity in the presence of 5 M urea. Upon drop dialysis against 0.2 M sodium phosphate buffer (pH 6.2), the purified pro-subtilisin in 5 M urea was processed to active subtilisin of which the N-terminal sequence and migration in SDS-polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E. This process was found to be very sensitive to the ionic strengths and anions used. Under the optimum conditions (dialysis against 0.5 M (NH4)2SO4 and 1 mM CaCl2 in 10 mM Tris-HCl buffer (pH 7.0) at 4 degrees C for 1 h), approximately 20% of pro-subtilisin E was converted to active subtilisin E. The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro-subtilisin E in which the active site was mutated (Asp32 to Asn) was unable to be processed under the optimum conditions. These results confirmed the previous hypothesis that the processing of pro-subtilisin occurs by an intramolecular, autoprocessing mechanism.

Ammonium Sulfate↗
PubMed1993

Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN' and Streptomyces subtilisin inhibitor.

An ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to > or = 11.5) of a tyrosyl residue of subtilisin BPN' in the interaction with carboxyls of SSI [Inouye et al. (1979) J. Biochem. 85, 1115-1126]. In the present paper, the tyrosyl residue in subtilisin BPN' and the carboxyls in SSI were identified by analyzing the difference spectrum using mutants of subtilisin BPN' and SSI: naturally occurring mutants and those prepared by site-directed and cassette mutagenesis. The difference spectrum disappeared on the binding of a mutant subtilisin BPN' of which Tyr104 was replaced by Phe (S-BPN'Y104F) and SSI at pH 9.8. The magnitude of the absorption difference was much smaller when subtilisin BPN' was bound with a mutant SSI of which both Glu67 and Asp68 were replaced by Gly than with the wild-type SSI. These lines of evidence indicated that the difference spectrum was caused by Tyr104 of subtilisin BPN' interacting with Glu67 and Asp68 of SSI. The binding of subtilisin BPN' and SSI is accompanied by an increase of tryptophan fluorescence, which is pH-dependent in the range of pH 7-11 [Uehara et al. (1978) J. Biochem. 84, 1195-1202]. In the present study, this pH-dependence of the fluorescence diminished when SSI bound with S-BPN'Y104F. This suggested that the fluorescence increase was due to Trp106 of subtilisin BPN' and was influenced by the ionization of Tyr104.

Amino Acids↗
PubMed1989

Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process.

Subtilisin E, an alkaline serine protease consisting of a single polypeptide chain of 275 amino acids is produced from a pre-pro-protein. The pre-sequence functions as the signal peptide for protein secretion across the membrane. Deletion of the pro-sequence yields mature but inactive subtilisin: the 77-amino acid pro-sequence must precede the mature subtilisin to guide the latter into an active conformation. Pro-subtilisin denatured in 6 M guanidine-HCl can be self-processed to the active enzyme intramolecularly, with concomitant cleavage of the pro-sequence, when dialysed against renaturing buffer. We have constructed an active-centre mutant of pro-subtilisin (Asp 32----Asn) which is not processed to active enzyme, unlike the wild-type pro-subtilisin, because intramolecular processing is prevented. Here we report an intermolecular pathway for the refolding of the inactive mature protein to an active enzyme in vitro with the aid of exogenously added pro-sequence. We establish conditions under which the mature inactive form, as well as acid-denatured subtilisins Carlsberg and BPN', can be renatured by the mutant pro-subtilisin.

Enzyme Activation↗
PubMed1979

[Subtilisin 72: a serine protease from Bac. subtilis strain 72 - an enzyme similar to subtilisin Carlsberg].

Subtilisin 72, a serine proteinase secreted by Bac. subtilis strain 72 was purified by covalent chromatography on Sepharose sorbent containing p-(omega-aminomethyl)phenylboronic acid as a ligand. The homogeneity of subtilisin 72 was confirmed by isoelectrofocusing in a thin layer of polyacrylamide gel (pl 8.6). The amino acid composition of this enzyme is different from that of other subtilisins, e. g. subtilisin Carlsberg. The N = terminal amino acid sequence of subtilisin 72 traced up to the 35th residue turned to be the same as that of subtilisin Carlsberg with the exception of the 21st (Tyr) and the 30th (Ile) residues. This very pronounced extent of homology shows that subtilisin 72 is very similar although not identical to subtilisin Carlsberg.

Amino Acid Sequence↗
PubMed1991

Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor.

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1979

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.

Amino Acids↗
PubMed1998

Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg.

The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (= sigma absolute value [(absolute value Fo) - (absolute value Fc)] / sigma (absolute value of Fo) of 15.1% using X-ray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid 'evolution' is able to accommodate considerable changes in sequence without substantial changes in property.

Amino Acid Sequence↗
PubMed1987

The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Structural analysis, subtilisin structure and interface geometry.

Triclinic crystals of the complex formed by eglin with subtilisin Carlsberg were analyzed by X-ray diffraction. The crystal and molecular structure of this complex was determined with data that extended to 0.12-nm resolution by a combination of Patterson search methods and isomorphous replacement techniques. Its structure was refined to a crystallographic R value of 0.178 (1.0-0.12 nm) using an energy-restraint least-squares procedure. The complete subtilisin molecule could be traced without ambiguity in the refined electron density. The eglin component, from which an amino-terminal segment is cleaved off, is only defined from Lys8I (i.e. the lysine residue 8 of the inhibitor) onwards. Per unit cell, 436 fixed solvent molecules and 2 calcium ions were located. In spite of 84 amino acid replacements and one deletion, subtilisin Carlsberg exhibits a very similar polypeptide fold to subtilisin BPN'. The root-mean-square deviations of all alpha-carbon atoms (excluding those at the deletion site) from models of subtilisin BPN' [Alden, R. A., Birktoft, J. J., Kraut, J., Robertus, J. D. & Wright, C. S. (1971) Biochem. Biophys. Res. Commun. 45, 337-344] and subtilisin Novo [Drenth, J., Hol, W. G. J., Jansonius, J. N. & Kockoek, R. (1972) Eur. J. Biochem. 25, 177-181] are 0.077 nm and 0.103 nm. Most of these deviations result from global shifts rather than changes of the local geometry. The single-residue deletion at position 56 affects only the surrounding conformation. Two sites of high electron density and close distances to surrounding oxygen ligands have been found in the Carlsberg enzyme which are probably occupied by calcium ions. Eglin consists of a twisted four-stranded beta-sheet flanked by an alpha-helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I-Asp46I, this loop is mainly stabilized by electrostatic/hydrogen bond interactions with the side chains of two arginine residues which project from the hydrophobic core [Bode, W., Papamokos, E., Musil, D., Seemüller, W. & Fritz, H. (1986) EMBO J. 5, 813-818]. The reactive site loop conformation resembles that found in other 'small' proteinase inhibitors. The scissile peptide bond is not cleaved but its carbonyl group is slightly distorted from planar geometry. Most of the intermolecular contacts are contributed by the nine residues of the reactive-site loop Gly40I-Arg48I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus.

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis strain DY was determined. This included automated sequence analysis of the whole molecule and its large fragments such as tryptic peptides obtained from the inactivated enzyme, peptides generated by cyanogen bromide, by o-iodosobenzoic acid and by hydroxylamine. The peptides were isolated by gel filtration and by reversed-phase high performance liquid chromatography. The amino-acid sequence of subtilisin DY was determined by overlapping the isolated peptides. It consists of 274 amino-acid residues, like that of subtilisin Carlsberg. By comparison with the structures of the subtilisins Carlsberg, amylosacchariticus and BPN' 32, 80 and 82 amino-acid substitutions were found, which are caused by 37, 102 and 106 nucleotide mutations, respectively. It was found also that 62.5% of the amino-acid residues in the molecules of these four subtilisins are identical with respect to kind and position of the residue, which suggests that these molecules have had a common ancestral precursor. The amino-acid replacement analysis of the four subtilisins leads to the conclusion that they have evolved almost independently.

Amino Acid Sequence↗
PubMed2001

Alkaline-resistance model of subtilisin ALP I, a novel alkaline subtilisin.

The alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of physicochemical methods. Subtilisin NAT (NAT), which exhibits no alkaline resistance, was examined as a control. ALP I gradually lost its activity, accompanied by protein degradation, but, on the contrary, Sendai was stable under alkaline conditions. CD spectral measurements at neutral and alkaline pH indicated no apparent differences between ALP I and Sendai. A significant difference was observed on measurement of fluorescence emission spectra of the tryptophan residues of ALP I that were exposed on the enzyme surface. The fluorescence intensity of ALP I was greatly reduced under alkaline conditions; moreover, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and optical activities of NAT were lost at high pH, indicating a lack of functional and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structure of the enzyme molecule.

Autolysis↗
PubMed1993

The elastolytic properties of subtilisin GX from alkalophilic Bacillus sp. strain 6644 provides a means of differentiation from other subtilisins.

A serine protease exhibiting high activity in alkaline media was purified from alkalophilic Bacillus strain GX6644. The enzyme, subtilisin GX, has a molecular weight of 25,000 and a pI greater than 9.5. The protease exhibited high elastolytic activity, but unlike most elastin hydrolyzing enzymes, elastin hydrolysis and binding were not inhibited by 0.1 M NaCl. The elastolytic properties of subtilisin GX together with its specificity toward amino acid phenyl esters functionally distinguishes this protease from other subtilisins. However, comparisons of the available amino-terminal sequence of subtilisin GX with subtilisins from alkalophilic and neutrophilic Bacillus species revealed extensive homology.

Amino Acid Sequence↗
PubMed2004

Stabilized variant of Streptomyces subtilisin inhibitor and its use in stabilizing subtilisin BPN'.

Protein protease inhibitors could potentially be used to stabilize proteases in commercial products such as liquid laundry detergents. However, many protein protease inhibitors are susceptible to hydrolysis inflicted by the protease. We have engineered Streptomyces subtilisin inhibitor (SSI) to resist proteolysis by adding an interchain disulfide bond and removing a subtilisin cleavage site at leucine 63. When these stabilizing changes were combined with changes to optimize the affinity for subtilisin, the resulting inhibitor provided complete protease stability for at least 5 months at 31 degrees C in a subtilisin-containing liquid laundry detergent and allowed full recovery of the subtilisin activity upon the dilution that occurs in a North American washing machine.

Amino Acid Sequence↗