Serine Protease PB92 Research Articles

Molecular dynamics studies of disulfide bonds for enhancing the stability of serine protease PB92

Employing innovative disulfide bond engineering and theoretical simulation, we introduced two bonds into PB92, enhancing thermal stability. The graphic illustrates these bonds, showcasing their crucial role in stabilizing the proteinase structure.

Improving the Thermostability of Serine Protease PB92 from Bacillus alcalophilus via Site-Directed Mutagenesis Based on Semi-Rational Design.

Proteases have been widely employed in many industrial processes. In this work, we aimed to improve the thermostability of the serine protease PB92 from Bacillus alcalophilus to meet the high-temperature requirements of biotechnological treatments. Eight mutation sites (N18, S97-S101, E110, and R143) were identified, and 21 mutants were constructed from B-factor comparison and multiple sequence alignment and expressed via Bacillus subtilis. Among them, fifteen mutants exhibited increased half-life (t1/2) values at 65 °C (1.13-31.61 times greater than that of the wild type). Based on the composite score of enzyme activity and thermostability, six complex mutants were implemented. The t1/2 values of these six complex mutants were 2.12-10.05 times greater than that of the wild type at 65 °C. In addition, structural analysis revealed that the increased thermal stability of complex mutants may be related to the formation of additional hydrophobic interactions due to increased hydrophobicity and the decreased flexibility of the structure. In brief, the thermal stability of the complex mutants N18L/R143L/S97A, N18L/R143L/S99L, and N18L/R143L/G100A was increased 4-fold, which reveals application potential in industry.

Gradient-purged isotope filter experiments for the detection of bound water in proteins

A combination of gradient-purged isotope-filtered NMR experiments is presented, which allows for the detection of long-lived bound water molecules in proteins. The discrimination of direct water–protein exchange from NOE effects between bound water and protein protons is achieved by NOE/ROE cancellation during the mixing time in one of the otherwise identical experiments. The method was applied successfully to 13 C/ 15 N -labelled serine protease PB92, and allowed for the identification of 22 protein–water NOEs in this 269-residue enzyme.

NMR structure calculation methods for large proteins Application of torsion angle dynamics and distance geometry/simulated annealing to the 269-residue protein serine protease PB92

Molecular dynamics in torsion angle space together with the well established combination of metric matrix distance geometry and simulated annealing in Cartesian space have been applied to the solution structure determination of serine protease PB92, a 269-residue monomeric protein from Bacillus alcalophilus. The input data set comprised distance restraints and a combination of distance and angular restraints derived from NMR data. A number of different modifications of the two calculation strategies were studied with respect to their convergence behaviour. The resulting structural ensembles were evaluated according to several criteria for protein structure quality. Improved protocols for both methods have been developed from these analyses. A comparison of the structures obtained with these protocols demonstrates the superior convergence behaviour of the torsion angle dynamics method. Although both methods are able to fold up the protein correctly, the torsion angle dynamics protocol scores slightly better with respect to measures of structural quality, especially when dihedral angle restraints are available. Torsion angle molecular dynamics presents a simple and robust method for the NMR structure calculations of large proteins.

NMR structure calculation methods for large proteins Application of torsion angle dynamics and distance geometry/ simulated annealing to the 269-residue protein serine protease PB92

NMR structure calculation methods for large proteins Application of torsion angle dynamics and distance geometry/ simulated annealing to the 269-residue protein serine protease PB92

Sensitivity Enhancement in the TROSY Experiment

A modification of the TROSY experiment which allows for sensitivity enhancement without introduction of additional delays is presented. The method relies on the combination of two orthogonal pathways and results in a signal-to-noise gain of2independent of the size of the molecule. The sensitivity enhanced TROSY scheme can be combined with gradient coherence selection. The method has been applied to the 269 residue serine protease PB92.

Structural and functional consequences of engineering the high alkaline serine protease PB92.

The ability to remove proteinaceous fabric stains made the alkaline proteases powerful tools in the hands of detergent manufacturers. The application of proteases in detergents requires that the proteases are stable and active in the presence of typical detergent ingredients such as surfactants, builders, bleaching agents, bleach activators, fillers, fabric softeners, various formulation aids, etc. To meet the high alkaline conditions in heavy duty powder detergents, extracellular Bacillus serine proteases have been extensively screened for good detergent stability and a high wash performance at this pH. In such a screening an alkalophilic Bacillus strain, PB92, was isolated1 which produces a serine protease with optimal performance in the pH range 10 to 12. The gene encoding this high-alkaline protease PB92 has been cloned and characterized2. The mature protease has a molecular weight of 26,900 and consists of 269 amino acids. A high sequence homology exists with protease Yab3 (83%) which is also produced by an alkalophilic Bacillus while moderate homology is found with the well known serine proteases from Bacillus amyloliquefaciens (subtilisin BPN’/NOVO, 59%)4,5, B. licheniformis (subtilisin Carlsberg, 60%)6,7, Tritirachium album Limber (proteinase-K, 34%)8,9, and Thermoactinomyces vulgaris (thermitase, 47%)10. In a recent review Siezen et al.11 proposed the name’subtilases’ for the group of serine proteases which share amino acid sequence homology with BPN’/NOVO in order to distinguish them from the (chymo)trypsin related serine proteases.

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

The 1H, 13C and 15N NMR resonances of serine protease PB92 have been assigned using 3D triple-resonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CHn carbons, all amide (NH and NH2) nitrogens and 99.2% of the amide and CHn protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405-411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.

1H, 13C and 15N NMR backbone assignments of the 269-residue serine protease PB92 from Bacillus alcalophilus

The (1)H, (13)C and (15)N NMR resonances of the backbone of serine protease PB92 have been assigned. This 269-residue protein is one of the largest monomeric proteins assigned so far. The amount and quality of information available suggest that even larger proteins could be assigned with present methods. Measured chemical shifts show excellent agreement with the secondary structure.

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket.

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)