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PubMed1994

Acylation of subtilisin with long fatty acyl residues affects its activity and thermostability in aqueous medium.

Subtilisin Carlsberg has been artificially hydrophobized by acylation with octanoyl or palmitoyl chlorides. Samples with several degrees of substitution were obtained. Hydrophobization facilitates in some cases the binding of synthetic or natural substrates. Furthermore, derivatized subtilisins show improved thermal stability (15-fold at 45 degrees C) in aqueous solution. As a result, octanoyl-subtilisin exhibits enhanced thermostability without losing biological activity.

Acylation↗

PubMed1983

Limited proteolysis of glycogen phosphorylase a by subtilisin BPN'.

The limited proteolysis of rabbit skeletal muscle phosphorylase a was undertaken with subtilisin BPN' immobilized to Sepharose 4B. The effect of substrates, activators and inhibitors of phosphorylase a was investigated by monitoring the changes in phosphorylase activity in the SDS gel electrophoretic pattern and in the 32P-content of 32P-labeled phosphorylase a. Phosphorylase a loses its activity upon subtilisin treatment. All ligands tested protect phosphorylase a activity against subtilisin action, probably by inducing structural changes in the tower loop of the enzyme. Glucose-6-P significantly accelerates [32P]peptide release from phosphorylase a through altering the structure of the N-terminal tail segment. The two subunits of dimeric phosphorylase a are held together by strong interactions--deduced from the correlation of the rate of proteolysis and the disappearance of catalytic activity.

Adenosine Monophosphate↗

PubMed1983

Limited proteolysis by subtilisin reveals structural differences between phosphorylase a and b.

The limited proteolysis of rabbit skeletal muscle phosphorylase a and b was studied with subtilisin BPN' immoblized to Sepharose 4B. The activity of phosphorylase b is nearly resistant to subtilisin under the conditions (pH 7.0, 30 degrees C) where phosphorylase a rapidly loses its activity. The pH profile of phosphorylase a and b digestion is different. Proteolytic fragments of mol. wt 70,000 and 30,000 generated from phosphorylase a, while mol. wt 80,000 and 70,000 generated from phosphorylase b can be detected by SDS gel electrophoresis. Addition of AMP to phosphorylase b favours a conformation similar to--but not identical with--phosphorylase a as recognised by subtilisin action.

Adenosine Monophosphate↗

PubMed1991

Refined crystal structures of subtilisin novo in complex with wild-type and two mutant eglins. Comparison with other serine proteinase inhibitor complexes.

The crystal structures of the complexes formed between subtilisin Novo and three inhibitors, eglin c, Arg45-eglin c and Lys53-eglin c have been determined using molecular replacement and difference Fourier techniques and refined at 2.4 A, 2.1 A, and 2.4 A resolution, respectively. The mutants Arg45-eglin c and Lys53-eglin c were constructed by site-directed mutagenesis in order to investigate the inhibitory specificity and stability of eglin c. Arg45-eglin became a potent trypsin inhibitor, in contrast to native eglin, which is an elastase inhibitor. This specificity change was rationalized by comparing the structures of Arg45-eglin and basic pancreatic trypsin inhibitor and their interactions with trypsin. The residue Arg53, which participates in a complex network of hydrogen bonds formed between the core and the binding loop of eglin c, was replaced with the shorter basic amino acid lysine in the mutant Lys53-eglin. Two hydrogen bonds with Thr44, located in the binding loop, can no longer be formed but are partially restored by a water molecule bound in the vicinity of Lys53. Eglin c in complexes with both subtilisin Novo and subtilisin Carlsberg was crystallized in two different space groups. Comparison of the complexes showed a rigid body rotation for the eglin c core of 11.5 degrees with respect to the enzyme, probably caused by different intermolecular contacts in both crystal forms.

Amino Acid Sequence↗

PubMed1993

Protein engineering of subtilisins to improve stability in detergent formulations.

Microbial proteases are used extensively in a large number of industrial processes and most importantly in detergent formulations facilitating the removal of proteinaceous stains. Site-directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. It is shown that in spite of significant structural homology between subtilisins subjected to protein engineering the effects of specific mutations can be quite different. Mutations that stabilize one subtilisin may destabilize another.

Amino Acid Sequence↗

PubMed1991

Purification and properties of a subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus.

A subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus were purified to homogeneity by conventional methods such as chromatography on DEAE-cellulose, gel filtration on Sephadex G-75, PAGE and affinity chromatography. The final preparations were homogeneous on PAGE. Their pI were 7.66 and 7.70, respectively. The dissociation constant of the complex of the inhibitor with subtilisin was 2.69.10(-10) M. Both the inhibitors were stable to heat, TCA and ethanol. The molecular weights of the subtilisin inhibitor and the associated trypsin inhibitor by gel filtration were 7500 and 8200, respectively.

Amino Acids↗

PubMed1986

Fusion of pro region of subtilisin to staphylococcal protein A and its secretion by Bacillus subtilis.

Subtilisin is synthesized as a preproenzyme in Bacillus subtilis. We fused that region of the subtilisin gene, (apr[BamP]), which encodes the signal sequence and pro region, to the mature gene sequence (spa) for a heterologous protein (staphylococcal protein A). B. subtilis cells harboring this gene fusion synthesized a fusion protein consisting of the signal and pro sequence of subtilisin fused to the protein A; the signal sequence was processed and a fusion protein (pro + protein A) was secreted into the growth medium.

Amino Acid Sequence↗

PubMed1995

Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis.

The cloning and sequence of the 5' untranslated region (5'-UTR) of the Bacillus licheniformis (Bl) 6816 subtilisin Carlsberg gene (subC) are reported here. The 5' and 3' ends of subC transcripts were characterized, and the promoter identified. Expression was studied using a fused lacZ reporter gene integrated into the chromosome of heterologous host Bacillus subtilis (Bs). beta Gal activities of mutants deleted within the promoter region identified a region which is required for stimulation by the transcriptional activator proteins, DegU and DegQ. This region is close to the transcription start point (tsp), and is adjacent to a sequence homologous to that involved in DegU/Q stimulation of the Bs subtilisin gene, aprE. Expression of subC in Bs was optimized by the use of heterologous promoter and by the deletion of UTR sequences predicted to be involved in secondary structures in the native subC mRNA. Sequence comparison with other subtilisin Carlsberg-type-encoding genes revealed a high degree of conservation of the entire 5'-UTR, including regulatory sequences and promoter, as well as part of the structural gene.

Amino Acid Sequence↗

PubMed2006

Obtaining high transesterification activity for subtilisin in ionic liquids.

It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF(6)]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg(-1) h(-1).

Chromatography, High Pressure Liquid↗

PubMed2004

Secreted subtilisin gene family in Trichophyton rubrum.

Secreted proteases constitute potential virulence factors of dermatophytes. A total of seven genes encoding putative serine proteases of the subtilisin family (SUB) were isolated in Trichophyton rubrum. Based on sequence data and intron-exon structure, a phylogenetic analysis of subtilisins from T. rubrum and other fungi revealed a presumed ancestral lineage comprising T. rubrum SUB2 and Aspergillus SUBs. All other SUBs (SUB1, SUB3-7) are dermatophyte-specific and have apparently emerged more recently, through successive gene duplication events. We showed that two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T. rubrum grown in a medium containing soy protein as a sole nitrogen source. Both recombinant enzymes produced in Pichia pastoris are highly active on keratin azure suggesting that these proteases play an important role in invasion of keratinised tissues by the fungus. The set of deduced amino acid sequences of T. rubrum SUB ORFs allowed the identification of orthologous Subs secreted by other dermatophyte species using proteolysis and mass spectrometry.

Cloning, Molecular↗

PubMed1988

Surface-mediated retention effects of subtilisin site-specific variants in cation-exchange chromatography.

Wild-type subtilisin and several site-specific variants were resolved on a strong cation-exchange column by isocratic elution and a series of sodium chloride concentrations. Changes in primary sequence at the protein surface have an observable effect on the chromatographic behavior of subtilisin. This supports the concept that three-dimensional structure determines which biopolymer surface residues are in position to interact with the stationary phase surface. The retention data fit the stoichiometric displacement model (SDM) of retention. Plots of ln k' vs. ln 1/[NaCl] yield values for the average number of ionic groups (Z) on the protein that interact with the support matrix. Application of the SDM to the chromatographic retention of the variants has uncovered an unusual phenomenon at the protein surface at low ionic strength. A SDM plot normally provides a linear relationship between ln k' and ln 1/[NaCl] with the slope corresponding to the Z number. This study revealed two lines differing in slope and intercept, indicating that the Z number of subtilisin changes at some intermediate ionic strength of the eluent. These results are attributed to some salt-induced protein surface event that triggers a change in structure. Chromatographic detection of this occurrence reflects the connection between the surface-mediated event and mobile phase ionic strength.

Amino Acids↗

PubMed1997

Gene from tropical Bacillus sphaericus encoding a protease closely related to subtilisins from Antarctic bacilli.

We undertook to identify the protease(s) involved in the in vivo degradation of the 100 kDa mosquitocidal toxin (Mtx) from Bacillus sphaericus SSII-1 and isolated a B. sphaericus SSII-1 gene flanked upstream by a typical Shine-Dalgarno ribosome binding site and downstream by a strong rho-independent transcription terminator. The predicted ORF encodes a 432 amino acid protein with significant homology throughout its sequence to two subtilisin-like serine proteases from the Antarctic psychrophilic (cold-adapted) bacilli, TA39 and TA41. The predicted N-terminal sequence suggests that the B. sphaericus protease is related to sfericase, a partially characterized serine protease from B. sphaericus. Only B. sphaericus strains which produce Mtx-degrading protease activity harbour the subtilisin-like protease gene, suggesting that this protease may be responsible for or contribute to the degradation of Mtx in B. sphaericus SSII-1. A 36-kDa protease with Mtx-degrading activity and similar properties to sfericase was also purified from sporulated cultures of B. sphaericus SSII-1. Further studies are needed to determine the relationship of this protease to sfericase and to the predicted product of the subtilisin-like serine protease gene.

Amino Acid Sequence↗

PubMed1998

Quantitative assay system for specific enzyme activity using antibody: the case of protease, subtilisin BPN'.

Assay on a high-quality microtiter plate was found to allow for quantitative analysis of bacterial serine protease, subtilisin BPN', and its mutant enzymes which had been genetically engineered to be adapted to low-temperatures (Taguchi et al., 1998. Appl. Environ. Microbiol. 64, 492-495), by using polyclonal antibody against subtilisin BPN'. The use of polyclonal antibody was crucial in normalizing the number of various different enzyme molecules in culture supernatant samples of recombinant strains of Bacillus subtilis, giving rise to the performance of specific activity assay of the enzymes. Relative activity of each mutant subtilisin BPN' to wild-type enzyme was estimated by monitoring the increased value of absorbance caused by enzymatic hydrolysis of a chromogenic substrate. The relative activity in each enzyme estimated by this method showed good coincidence with that estimated by kinetic parameters, kcat/K(m) of the purified enzymes. We termed the system as 'ABEA' (antibody-bound enzyme assay). The efficient ABEA system developed here would be useful for the determination of specific activity of other enzymes of interest and provide us versatile applications in the field of evolutionary engineering.

Animals↗

PubMed2001

New subtilisin-like collagenase from leaves of common plantain.

A new subtilisin-like proteinase hydrolyzing chromogenic peptide substrate Glp-Ala-Ala-Leu-p-nitroanilide optimally at pH 8.1 was found in common plantain leaves. The protease named plantagolisin was isolated by ammonium sulfate precipitation of the leaves' extract followed by affinity chromatography on bacitracin-Sepharose and ion-exchange chromatography on Mono Q in FPLC regime. Its molecular mass is 19000 Da and pI 5.0. pH-stability range is 7-10 in the presence of 2 mM Ca(2+), temperature optimum is 40 degrees C. The substrate specificity of subtilase towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases: proteinase from leaves of the sunflower and taraxalisin. Besides, the proteinase is able to hydrolyze substrates with Pro in P(1) position. The enzyme hydrolyzes collagen. alpha and beta chains are hydrolyzed simultaneously in parallel; there are only low-molecular-mass hydrolysis products in the sample after 2 h of incubation. Pure serine proteinase was inactivated by specific serine proteinases inhibitors: diisopropylfluorophosphate, phenylmethylsulfonyl fluoride and Hg(2+). The plantagolisin N-terminal sequence ESNSEQETQTESGPGTAFL-, traced for 19 residues, revealed 37% homology with that of subtilisin from yeast Schizosaccharomyces pombe.

Amino Acid Sequence↗

PubMed1996

Subtilisin-catalyzed religation of proteolyzed hen egg-white lysozyme: investigation of the role of disulfides.

BACKGROUND: The use of proteases to form, instead of break, peptide bonds has expanded the repertoire of techniques available for protein semisynthesis. Several groups have previously reported the use of proteases in aqueous-organic solvents to form single amide bonds within proteins, but low yields and lengthy reaction times make this an impractical approach to protein synthesis. We recently found that proteolyzed triose phosphate isomerase can be re-ligated rapidly and efficiently by subtilisin, in mixed aqueous-organic solvent systems. RESULTS: We now report the use of subtilisin to resynthesize hen egg-white lysozyme from a mixture of its proteolyzed fragments in high yield and with rapid reaction times. This enzymatic religation can also be achieved after reduction of the four disulfide bonds present in lysozyme, with the same efficiency as that observed for the disulfide-containing proteolysis mixture. CONCLUSIONS: For egg-white lysozyme, the subtilisin religation reaction can be used to re-synthesize a proteolyzed protein even after reduction of disulfide bonds. The utility of this reaction in more generalized protein semisynthesis reactions is currently being explored.

Animals↗

PubMed1997

Improving the activity of immobilized subtilisin by site-specific attachment to surfaces.

Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors. The protease subtilisin BPN' was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties. To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis. This cysteine residue was designed to be away from the active site of the enzyme. The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports. This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase. Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization.

Bacillus↗

PubMed1992

Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching.

Subtilisins are serine endopeptidases with an extended binding cleft comprising at least eight binding subsites. Interestingly, subsites distant from the scissile bond play a dominant role in determining the specificity of the enzymes. The development of internally quenched fluorogenic substrates, which allow polypeptides of more than 11 amino acids to be inserted between the donor and the acceptor, has rendered it possible to perform a highly systematic mapping of the individual subsites of the active sites of subtilisin BPN' from Bacillus amyloliquefaciens and Savinase from Bacillus lentus. For each enzyme, the eight positions S5-S'3 were characterized by determination of kcat/KM values for the hydrolysis of substrates in which the amino acids were systematically varied. The results emphasize that in both subtilisin BPN' and Savinase interactions between substrate and S4 and S1 are very important. However, it is apparent that interactions between other subsites and the substrate exert a significant influence on the substrate preference. The results are rationalized on the basis of the structural data available for the two enzymes.

Amino Acid Sequence↗

PubMed1991

Neutron structure of subtilisin BPN': effects of chemical environment on hydrogen-bonding geometries and the pattern of hydrogen-deuterium exchange in secondary structure elements.

The neutron structure of subtilisin BPN' has been refined and analyzed at 2.0-A resolution. The structure studied was a mutant variant of subtilisin, Met222----Gln, and was used because large, uninhibited crystals could be grown, which was not the case for the native molecule. Comparison of the structure with that of the native molecule indicated that the two structures are essentially the same. Using the capability of the neutron method to locate hydrogen and deuterium atoms, the protonation states of the six histidine residues were assigned. The active site histidine, His64, was found to be neutral at the pH of the analysis (pH 6.1). This group has an unexpectedly low pKa compared to assignments made by other techniques. The altered pKa of the group could result from electrostatic effects of other molecules in the crystal lattice. The dihedral conformations of a majority of the hydroxyl rotors were assigned. The preferred orientation was trans (180 degrees) with the other two low-energy conformers (60 degrees, 300 degrees) about equally populated. For the serines, about 21% of the hydroxyls act exclusively as H-bond acceptors and 37% as H-bond donors, and in 42% the group functions as both. The experimentally observed dihedral conformations were compared to predicted conformations based on calculated energy criteria and showed a strong correspondence. Deviation from low-energy states could usually be explained by local electrostatic effects. The hydrogen exchange pattern of subtilisin identified the beta-sheet and alpha-helix secondary structure elements to be the most resistant to exchange. Fifty-five percent of the peptide amide hydrogens were fully exchanged, 15% unexchanged, and 30% partially exchanged. The largest concentration of unexchanged sites was in the seven-stranded parallel beta-sheet, in which there were 11 fully protected groups. Little correlation was found between H-bond length and angle and a peptide group's susceptibility toward exchange. Of the five alpha-helices the most protected from exchange is the one defined by residues 224-236. The pattern of exchange identifies regions in this helix where the H-bonding regularity is disrupted.

Binding Sites↗