Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

The Autocatalytic Cleavage Domain Is Not Required for the Activity of ScpC, a Virulence Protease from Streptococcus pyogenes: A Structural Insight.

Group A Streptococcus (GAS, or Streptococcus pyogenes) is a leading human bacterial pathogen with diverse clinical manifestations, ranging from mild to life-threatening and to severe immune sequela. These diseases, combined, account for more than half a million deaths per year, globally. To accomplish its vast pathogenic potential, GAS expresses a multitude of virulent proteins, including the pivotal virulence factor ScpC. ScpC is a narrow-range surface-exposed subtilisin-like serine protease that cleaves the last 14 C-terminal amino acids of interleukin 8 (IL-8 or CXCL8) and impairs essential IL-8 signaling processes. As a result, neutrophil migration, bacterial killing, and the formation of neutrophil extracellular traps are strongly impaired. Also, ScpC has been identified as a potential vaccine candidate. ScpC undergoes an autocatalytic cleavage between Gln244 and Ser245, resulting in two polypeptide chains that assemble together forming the active protease. Previously, we reported that the region harboring the autocatalytic cleavage site, stretching from Gln213 to Asp272, is completely disordered. Here, we show that a deletion mutant (ScpCΔ60) of this region forms a single polypeptide chain, whose crystal structure we determined at 2.9 Å resolution. Moreover, we show that ScpCΔ60 is an active protease capable of cleaving its substrate IL-8 in a manner comparable to that of the wild type. These studies improve our understanding of the proteolytic activity of ScpC.

Structural insights into the oligomerization of FtsH periplasmic domain from Thermotoga maritima.

Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. The transmembrane protein, FtsH, is the only known ATP-dependent protease responsible for this task. The mechanisms by which FtsH recognizes, unfolds, translocates, and proteolyzes its substrates remain unclear. The structure and function of the ATPase and protease domains of FtsH have been previously characterized while the role of the FtsH periplasmic domain has not clearly identified. Here, we report the 1.5-1.95 Å resolution crystal structures of the Thermotoga maritima FtsH periplasmic domain (tmPD) and describe the dynamic features of tmPD oligomerization.

Effect of the viral protease on the dynamics of bacteriophage HK97 maturation intermediates characterized by variance analysis of cryo EM particle ensembles.

Cryo EM structures of maturation-intermediate Prohead I of bacteriophage HK97 with (PhI(Pro+)) and without (PhI(Pro-)) the viral protease packaged have been reported (Veesler et al., 2014). In spite of PhI(Pro+) containing an additional ∼ 100 × 24 kD of protein, the two structures appeared identical although the two particles have substantially different biochemical properties, e.g., PhI(Pro-) is less stable to disassembly conditions such as urea. Here the same cryo EM images are used to characterize the spatial heterogeneity of the particles at 17Å resolution by variance analysis and show that PhI(Pro-) has roughly twice the standard deviation of PhI(Pro+). Furthermore, the greatest differences in standard deviation are present in the region where the δ-domain, not seen in X-ray crystallographic structures or fully seen in cryo EM, is expected to be located. Thus presence of the protease appears to stabilize the δ-domain which the protease will eventually digest.

Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within biomimetically generated silicate nanospheres.

BACKGROUND: Dendrimers are highly branched synthetic macromolecules with a globular shape. They have been successfully used for generation of nanospheres at mild conditions via biomimetic silicification. Encapsulation of enzyme molecules within these nanospheres during their synthesis is a promising method for rapid and efficient entrapment of several enzymes. However, encapsulation of proteolytic enzymes has been rarely done via biomimetic silicification. As well, the operational stability of encapsulated enzyme has not been systematically reported. METHODS: A proteolytic enzyme, either alpha-Chymotrypsin or a fungal protease from Aspergilus Oryzea was encapsulated along with iron oxide nanoparticles within particles yielded via biomimetic silicification of different generations of polyamidoamine (PAMAM) dendrimers. Stability of encapsulated enzyme was compared to that of free enzyme during storage at room temperature. As well, their thermal and ultrasonic stabilities were measured. Scanning electron microscopy, transmission electron microscopy and optical microscopy were used to investigate the morphology of nanospheres. RESULTS: Determination of encapsulation efficiency revealed that approximately 85% of fungal protease with concentration 1.4mg mL(-1) stock solution was immobilized within particles yielded by generation 0. Based on microscopic images the generated particles interconnected with each other and had spherical morphologies independent of generation. Kinetic analysis of encapsulated fungal protease demonstrated that Mechaelis-Menten constant (K(m)) slightly increased. CONCLUSION: PAMAM dendrimer generation 0 could be effectively used for rapid encapsulation of a fungal protease from Aspegilus Oryzae. GENERAL SIGNIFICANCE: Encapsulation significantly enhances the thermal and ultrasonic stabilities of enzymes, suggesting a range of diverse applications for them.

Structural and biochemical characterization of a novel thermophilic Coh01147 protease.

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/ß sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function.

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

A study on the protease activity and structure of pepsin in the presence of atenolol and diltiazem.

Pepsin, as the main protease of the stomach, plays an important role in the digestion of food proteins into smaller peptides and performs about 20% of the digestive function. The role of pepsin in the development of gastrointestinal ulcers has also been studied for many years. Edible drugs that enter the body through the gastrointestinal tract will interact with this enzyme as one of the first targets. Continuous and long-term usage of some drugs will cause chronic contact of the drug with this protein, and as a result, the structure and function of pepsin may be affected. Therefore, the possible effect of atenolol and diltiazem on the structure and activity of pepsin was studied. The interaction of drugs with pepsin was evaluated using various experimental methods including UV-Visible spectroscopy, fluorescence spectroscopy, FTIR and enzymatic activity along with computational approaches. It was showed that after binding of atenolol and diltiazem to pepsin, the inherent fluorescence of the protein is quenched. Determination of the thermodynamic parameters of interactions between atenolol and diltiazem with pepsin indicates that the major forces in the formation of the protein-drug complexes are hydrophobic forces and also atenolol has a stronger protein bonding than diltiazem. Additional tests also show that the protease activity of pepsin, decreases and increases in the presence of atenolol and diltiazem, respectively. Investigation of the FTIR spectrum of the protein in the presence and absence of atenolol and diltiazem show that in the presence of atenolol the structure of protein has slightly changed. Molecular modeling studies, in agreement with the experimental results, confirm the binding of atenolol and diltiazem to the enzyme pepsin and show that the drugs are bind close to the active site of the enzyme. Finally, from experimental and computational results, it can be concluded that atenolol and diltiazem interact with the pepsin and change its structure and protease activity.

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme.

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.

Structural comparison strengthens the higher-order classification of proteases related to chymotrypsin.

Specific cleavage of proteins by proteases is essential for several cellular, physiological, and viral processes. Chymotrypsin-related proteases that form the PA clan in the MEROPS classification of proteases is one of the largest and most diverse group of proteases. The PA clan comprises serine proteases from bacteria, eukaryotes, archaea, and viruses and chymotrypsin-related cysteine proteases from positive-strand RNA viruses. Despite low amino acid sequence identity, all PA clan proteases share a conserved double ß-barrel structure. Using an automated structure-based hierarchical clustering method, we identified a common structural core of 72 amino acid residues for 143 PA clan proteases that represent 12 protein families and 11 subfamilies. The identified core is located around the catalytic site between the two ß-barrels and resembles the structures of the smallest PA clan proteases. We constructed a structure-based distance tree derived from the properties of the identified common core. Our structure-based analyses support the current classification of these proteases at the subfamily level and largely at the family level. Structural alignment and structure-based distance trees could thus be used for directing objective classification of PA clan proteases and to strengthen their higher order classification. Our results also indicate that the PA clan proteases of positive-strand RNA viruses are related to cellular heat-shock proteases, which suggests that the exchange of protease genes between viruses and cells might have occurred more than once.

Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome.

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

Source of high pathogenicity of an avian influenza virus H5N1: why H5 is better cleaved by furin.

The origin of the high pathogenicity of an emerging avian influenza H5N1 due to the -RRRKK- insertion at the cleavage loop of the hemagglutinin H5, was studied using the molecular dynamics technique, in comparison with those of the noninserted H5 and H3 bound to the furin (FR) active site. The cleavage loop of the highly pathogenic H5 was found to bind strongly to the FR cavity, serving as a conformation suitable for the proteolytic reaction. With this configuration, the appropriate interatomic distances were found for all three reaction centers of the enzyme-substrate complex: the arrangement of the catalytic triad, attachment of the catalytic Ser(368) to the reactive S1-Arg, and formation of the oxyanion hole. Experimentally, the--RRRKK--insertion was also found to increase in cleavage of hemagglutinin by FR. The simulated data provide a clear answer to the question of why inserted H5 is better cleaved by FR than the other subtypes, explaining the high pathogenicity of avian influenza H5N1.

Microseconds dynamics simulations of the outer-membrane protease T.

Conformational fluctuations of enzymes may play an important role for substrate recognition and/or catalysis, as it has been suggested in the case of the protease enzymatic superfamily. Unfortunately, theoretically addressing this issue is a problem of formidable complexity, as the number of the involved degrees of freedom is enormous: indeed, the biological function of a protein depends, in principle, on all its atoms and on the surrounding water molecules. Here we investigated a membrane protease enzyme, the OmpT from Escherichia coli, by a hybrid molecular mechanics/coarse-grained approach, in which the active site is treated with the GROMOS force field, whereas the protein scaffold is described with a Go-model. The method has been previously tested against results obtained with all-atom simulations. Our results show that the large-scale motions and fluctuations of the electric field in the microsecond timescale may impact on the biological function and suggest that OmpT employs the same catalytic strategy as aspartic proteases. Such a conclusion cannot be drawn within the 10- to 100-ns timescale typical of current molecular dynamics simulations. In addition, our studies provide a structural explanation for the drop in the catalytic activity of two known mutants (S99A and H212A), suggesting that the coarse-grained approach is a fast and reliable tool for providing structure/function relationships for both wild-type OmpT and mutants.

The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1.

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.

Homology in structural organization between E. coli ClpAP protease and the eukaryotic 26 S proteasome.

Energy-dependent protein degradation is carried out by large multimeric protein complexes such as the proteasomes of eukaryotic and archaeal cells and the ATP-dependent proteases of eubacterial cells. Clp protease, a major multicomponent protease of Escherichia coli, consists of a proteolytic component, ClpP, in association with an ATP-hydrolyzing, chaperonin-like component, ClpA. To provide a structural basis for understanding the regulation and mechanism of action of Clp protease, we have used negative staining electron microscopy and image analysis to examine ClpA and ClpP separately, as well as active ClpAP complexes. Digitized images of ClpP and ClpA were analyzed using a novel algorithm designed to detect rotational symmetries. ClpP is composed of two rings of seven subunits superimposed in bipolar fashion along the axis of rotational symmetry. This structure is similar to that formed by the beta subunits of the eukaryotic and archaeal proteasomes. In the presence of MgATP, ClpA forms an oligomer with 6-fold symmetry when viewed en face. Side views of ClpA indicate that the subunits are bilobed with the respective domains forming two stacked rings. ClpAP complexes contain a tetradecamer of ClpP flanked at one or both ends with a hexamer of ClpA, resulting in a symmetry mismatch between the axially aligned molecules. Our findings demonstrate that, despite the lack of sequence similarity between ClpAP and proteasomes, these multimeric proteases nevertheless have a profound similarity in their underlying architecture that may reflect a common mechanism of action.

Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome.

The COP9 signalosome is involved in signal transduction, whereas the 26 S proteasome lid is a regulatory subcomplex of the 26 S proteasome responsible for degradation of ubiquitinated proteins. COP9 signalosome and lid possess significant sequence homologies among their eight core subunits and are likely derived from a common ancestor. Surprisingly, from our two-dimensional electron microscopy data, a common architectural plan for the two complexes could not be deduced. None-the-less, the two particles have structural features in common. Both COP9 signalosome and lid lack any symmetry in subunit arrangement and exhibit a central groove, possibly qualified for scaffolding functions.Filter-binding assays with recombinant COP9 signalosome components revealed a multitude of subunit-subunit interactions, supporting the asymmetrical appearance of the complex in electron microscopy. On the basis of two-dimensional images and subunit interaction studies, a first architectural model of COP9 signalosome was created. The fact that four distinct classes of particle views were identified and that only 50 % of the selected particles could be classified indicates a high degree of heterogeneity in electron microscopic images. Different orientations with respect to the viewing axis and conformational variety, presumably due to different grades of phosphorylation, are possible reasons for the heterogeneous appearance of the complex. Our biochemical data show that recombinant COP9 signalosome subunits 2 and 7 are phosphorylated by the associated kinase activity. The modification of COP9 signalosome subunit 2 might be essential for c-Jun phosphorylation. Dephosphorylation does not inactivate the associated kinase activity. Although substrate phosphorylation by COP9 signalosome is significantly decreased by lambda protein phosphatase treatment, "autophosphorylation" is increased.

Mining frequent patterns in protein structures: a study of protease families.

MOTIVATION: Analysis of protein sequence and structure databases usually reveal frequent patterns (FP) associated with biological function. Data mining techniques generally consider the physicochemical and structural properties of amino acids and their microenvironment in the folded structures. Dynamics is not usually considered, although proteins are not static, and their function relates to conformational mobility in many cases. RESULTS: This work describes a novel unsupervised learning approach to discover FPs in the protein families, based on biochemical, geometric and dynamic features. Without any prior knowledge of functional motifs, the method discovers the FPs for each type of amino acid and identifies the conserved residues in three protease subfamilies; chymotrypsin and subtilisin subfamilies of serine proteases and papain subfamily of cysteine proteases. The catalytic triad residues are distinguished by their strong spatial coupling (high interconnectivity) to other conserved residues. Although the spatial arrangements of the catalytic residues in the two subfamilies of serine proteases are similar, their FPs are found to be quite different. The present approach appears to be a promising tool for detecting functional patterns in rapidly growing structure databases and providing insights in to the relationship among protein structure, dynamics and function. AVAILABILITY: Available upon request from the authors.

Proteasome inhibitors in the treatment of B-cell malignancies.

The proteasome, which plays a pivotal role in the control of many cell cycle-regulatory processes, has become the focus of new approaches to the treatment of cancer, including B-cell malignancies, and the first proteasome inhibitor, bortezomib (VELCADE; formerly PS-341), has entered clinical trials. The proteasome controls the stability of numerous proteins that regulate progression through the cell cycle and apoptosis, such as cyclins, cyclin-dependent kinases, tumor suppressors, and the nuclear factor-kB. By altering the stability or activity of these proteins, proteasome inhibitors sensitize malignant cells to apoptosis. Bortezomib is a dipeptidyl boronic acid proteasome inhibitor that effectively and specifically inhibits proteasome activity. In preclinical studies, bortezomib and other proteasome inhibitors have shown activity against a variety of B-cell malignancies, including multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, and Hodgkin's lymphoma. These agents can induce apoptosis and sensitize tumor cells to radiation or chemotherapy. Based on these findings, phase I clinical trials were conducted with bortezomib in various solid and hematologic malignancies. In these studies, bortezomib was generally well tolerated with manageable toxicities. Phase II trials have been initiated for relapsed and refractory multiple myeloma, refractory chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Preliminary data from the multiple myeloma phase II study indicate that a significant number of patients responded to therapy or exhibited stable disease and that the drug had manageable toxicities. These findings, along with extensive preclinical data, suggest that bortezomib and other proteasome inhibitors may have far-reaching potential in the treatment of various cancers, including B-cell malignancies.

Studies on PVA pectin cryogels containing crosslinked enzyme aggregates of keratinase.

Polyvinyl alcohol-pectin (PVA-P) films containing enrofloxacin and keratinase were developed to treat wounds and scars produced by burns and skin injuries. However, in order to prevent enzyme inactivation at the interface between the patch and the scars, crosslinked enzyme aggregates (CLEAs) from a crude extract of keratinase produced by Paecilomyces lilacinus (LPSC#876) were synthesized by precipitation with acetone and crosslinking with glutaraldehyde. Soluble vs. CLEA keratinase (K-CLEA) activities were tested in 59% (v/v) hydrophobic (isobutanol and n-hexane) and hydrophilic (acetone and dimethylsulfoxide) solvents mixtures. K-CLEA activity was 1.4, 1.7 and 6.6 times higher in acetone, n-hexane and isobutanol than the soluble enzyme at 37 °C after 1 h of incubation, respectively. K-CLEA showed at least 45% of enzyme residual activity in the 40-65 °C range, meanwhile the soluble biocatalyst was fully inactivated at 65 °C after 1h incubation. Also, the soluble enzyme was completely inactivated after 12 h at pH 7.4 and 45 °C, even though K-CLEA retained full activity. The soluble keratinase was completely inactivated at 37 °C after storage in buffer solution (pH 7.4) for 2 months, meanwhile K-CLEAs kept 51% of their activity. K-CLEA loaded into polyvinyl alcohol (PVA) and PVA-P cryogels showed six times lower release rate compared to the soluble keratinase at skin pH (5.5). Small angle X-ray scattering (SAXS) analysis showed that K-CLEA bound to pectin rather than to PVA in the PVA-P matrix.

In situ synthesis of porous silica nanoparticles for covalent immobilization of enzymes.

A simple method is used to covalently encapsulate enzymes in silica nanoparticles. The encapsulation is highlighted by the high enzyme loading and porous channels that provide efficient diffusion for small substrate and product molecules while preventing protease degradation.