Probing the collective vibrational dynamics of a protein in liquid water by terahertz absorption spectroscopy

Biological polymers are expected to exhibit functionally relevant, global, and subglobal collective modes in the terahertz (THz) frequency range (i.e., picosecond timescale). In an effort to monitor these collective motions, we have experimentally determined the absorption spectrum of solvated bovine serum albumin (BSA) from 0.3 to 3.72 THz (10-124 cm(-1)). We successfully extract the terahertz molar absorption of the solvated BSA from the much stronger attenuation of water and observe in the solvated protein a dense, overlapping spectrum of vibrational modes that increases monotonically with increasing frequency. We see no evidence of distinct, strong, spectral features, suggesting that no specific collective vibrations dominate the protein's spectrum of motions, consistent with the predictions of molecular dynamics simulations and normal mode analyses of a range of small proteins. The shape of the observed spectrum resembles the ideal quadratic spectral density expected for a disordered ionic solid, indicating that the terahertz normal mode density of the solvated BSA may be modeled, to first order, as that of a three-dimensional elastic nanoparticle with an aperiodic charge distribution. Nevertheless, there are important detailed departures from that of a disordered inorganic solid or the normal mode densities predicted for several smaller proteins. These departures are presumably the spectral features arising from the unique molecular details of the solvated BSA. The techniques used here and measurements have the potential to experimentally confront theoretical calculations on a frequency scale that is important for macromolecular motions in a biologically relevant water environment.     

Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV–vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60 °C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80 °C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50 kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90 °C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.     

Effects of temperature on ultrasound-assisted tryptic protein digestion

The effects of temperature on ultrasound-assisted tryptic protein digestion were comprehensively investigated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Three standard proteins, cytochrome c, myoglobin, and bovine serum albumin, were digested at 4 °C (ice), room temperature (20–25), 37, and 55 °C for 0 s, 30 s, 1 min, and 5 min, in an ultrasonic bath. We found that the number of identified peptides generally increased with increasing temperature or digestion time. Compared with conventional overnight digestion at 37 °C without ultrasonication, digestions performed under ultrasonication generally produced more peptides under most of the above listed conditions, mainly due to miscleaved peptides. Tryptic digestions were also performed under all the conditions evaluated without using ultrasound, where the most significant improvement with the application of ultrasound in terms of sequence coverage and the number of identified peptides was observed at 4 °C, followed by room temperature, and 37 °C, while no improvement was observed at 55 °C with the application of ultrasound, which may be due to the fact that the current experiments were performed in an ultrasonic bath.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Different pressure–temperature behavior of the structured and unstructured regions of titin

Contrary to the classical view, according to which all proteins adopt a specific folded conformation necessary for their function, intrinsically unstructured proteins (IUPs) display random-coil-like conformation under physiological conditions. We compared the structured and unstructured domains from titin, a giant protein responsible for striated-muscle elasticity. A 171-residue-long fragment (polyE) of the disordered PEVK domain, and an Ig domain (I27) with ordered structure were investigated. FTIR (Fourier transform infrared) and fluorescence spectroscopy combined with a diamond anvil cell were used for investigation of the secondary structures under wide range of pressure and temperature. PolyE preserves its disordered characteristics across the entire range of investigated pressure (0–16 kbar), temperature (0–100 °C), pD (3–10.5) and different solvent conditions. The detailed temperature–pressure phase diagram of titin I27 was determined. At 30 °C, increasing pressure unfolds titin I27 in one step at 10.5 kbar. Increasing temperature at atmospheric pressure results in two transitions. At 50 °C the secondary structure is loosened and the protein transforms into a molten-globule state. At 65 °C the protein completely unfolds. Unfolding is followed by aggregation at ambient pressure. Moderate pressures (> 2 kbar), however, can prevent the protein from aggregation. Our experiments in wide range of physical parameters revealed four different structures for I27, while the unstructured character of the PEVK fragment is insensitive to these parameters.     

Dipeptidyl peptidase IV activity in commercial solutions of human serum albumin

Due to the heterogeneous nature of commercial human serum albumin (cHSA), other components, such as the protease dipeptidyl peptidase IV (DPP-IV), possibly contribute to the therapeutic effect of cHSA. Here, we provide evidence for the first time that DPP-IV activity contributes to the formation of aspartate–alanine diketopiperazine (DA-DKP), a known immunomodulatory molecule from the N terminus of human albumin. cHSA was assayed for DPP-IV activity using a specific DPP-IV substrate and inhibitor. DPP-IV activity was assayed at 37 and 60 °C because cHSA solutions are pasteurized at 60 °C. DPP-IV activity in cHSA was compared with other sources of albumin such as a recombinant albumin (rHSA). In addition, the production of DA-DKP was measured by negative electrospray ionization/liquid chromatography mass spectrometry (ESI⁻/LCMS). Significant levels of DPP-IV activity were present in cHSA. This activity was abolished using a specific DPP-IV inhibitor. Fully 70 to 80% DPP-IV activity remained at 60 °C compared with the 37 °C incubate. No DPP-IV activity was present in rHSA, suggesting that DPP-IV activity is present only in HSA produced using the Cohn fractionation process. The formation of DA-DKP at 60 °C was observed with the DPP-IV inhibitor significantly decreasing this formation. DPP-IV activity in cHSA results in the production of DA-DKP, which could account for some of the clinical effects of cHSA.     

Pressure and Temperature Dependence of Growth and Morphology of Escherichia coli: Experiments and Stochastic Model

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1–400 atm) and temperature (23–40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.     

Temperature and pH-Dependent Supramolecular Self-Assembly of Amelogenin Molecules: A Dynamic Light-Scattering Analysis

Evidence for the molecular self-assembly of amelogenin proteins to form quasi-spherical particles (“nanospheres”) in solution, bothin vitroandin vivo,has recently been documented. A particle-size distribution analysis of dynamic light-scattering data was undertaken to investigate the influence of temperature on this molecular self-assembly process at three different pH's. The long-term objective was to correlate these observations to the unusual physiochemical characteristics of the protein, to improve understanding of the molecular mechanisms involved in the generation of amelogenin “nanospheres” and understanding of their putative relation to amelogenin functionin vivo. We analyzed data using two different algorithms: Dynamics and DynaLS. It was found that at pH 8, in a temperature range between 5 and 25°C, the size of the recombinant amelogenin nanospheres is monodisperse, giving rise to particles of 15–18 nm in hydrodynamic radius. However, heterogeneous distribution of particle size was observed at temperature ranges between 27 and 35°C, becoming monodisperse again with larger particles (60–70 nm) after the temperature was elevated to 37–40°C. We interpret these results to suggest that amelogenin molecular self-association possesses a second stage assembly process at temperatures of 30–35°C, creating larger entities which apparently are structured and stable at 37–40°C. The effect of pH on the size of amelogenin “aggregates” was much more noticeable at 37°C compared to that at 25°C. This observation suggests that at physiological temperature (i.e., 37°C) amelogenin molecular self-assembly is extremely sensitive to pH changes. This finding supports the notion that local pH changes in the microenvironment of the enamel extracellular matrix may play critical roles in controlling the structural organization of the organic matrix framework.     

Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium–HCl,urea, and pH

The stability of bovine lactoperoxidase to denaturation by guanidinium–HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near T_m is controlled by kinetics. The values for the thermal transition, T_m, at slow heating scan rate were 66.8, 61.1, and 47.2 °C in the presence of 0.5, 1, and 2 M guanidinium–HCl, respectively. The extrapolated value for T_m in the absence of guanidinium–HCl is 73.7 °C, compared with 70.2 °C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, C_p, at T_m and E_a for the thermal transition decrease under conditions where T_m is lowered. At a given concentration, urea is less effective than guanidinium–HCl in reducing T_m, but urea reduces C_p relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 °C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164–1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.     

Effects of heating at neutral and acid pH on the structure of β-lactoglobulin A revealed by differential scanning calorimetry and circular dichroism spectroscopy

The structural change of β-lactoglobulin A (βLG A) on heating was measured at pH 3.0 and 7.5 with UV absorption difference spectra, differential scanning calorimetry (DSC), and circular dichroism (CD). At pH 3.0, βLG A showed a reversible structural change by heating at 80 °C, while an irreversible change was observed and molecular aggregates of βLG were formed by heating at 95 °C. DSC analysis of βLG A gave endothermic peaks at 75 °C and 90 °C at pH 7.5, and 90 °C at pH 3.0. At pH 7.5, βLG A modified with N-ethylmaleimide (NEM-βLG A) gave two endothermic peaks: at 72 °C and 90 °C. CD spectra of βLG A heated at various temperatures and pHs were measured and the spectra at pH 3.0 and 7.5 were not changed by heating to 95 °C and 80 °C, respectively. Unheated NEM-βLG A gave a spectrum similar to that of heated βLG A, suggesting that the secondary structure was changed by NEM treatment.     

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH 7.4 (pI = 7–8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 °C) and molar enthalpy (146 kcal M^− 1) values increasing to 78 °C and 163 kcal M^− 1 at 10 μM holo-FBP, while those of apo-FBP were 55 °C and 105 kcal M^− 1. Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10 μM) in DLS after a step-wise rise in temperature to 78 °C ≈ Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 °C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.     

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I′ band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 °C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 °C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 °C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.     

Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb–A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K_D ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP–sdAb gave a yield of approximately 100 mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb–A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1 mg/ml for 1 h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1 h at 90 °C. The PfuMBP–sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb–A5 both as a capture reagent and as a detection reagent.     

Red chlorophyll excitation dynamics in Arthrospira platensis photosystem I trimeric complexes as studied by femtosecond transient absorption spectroscopy

Femtosecond absorption spectroscopy was applied to study for the first time excitation dynamics in isolated photosystem I trimers from Arthrospira platensis, which display extremely long-wavelength absorption peaks. Pump–probe spectra observed at 77 K in the timescale of dozens of picoseconds upon 70-fs excitation revealed two maxima near 710 and 730 nm, which correspond to red chlorophyll forms. Bleaching at 680 nm developed in ∼200 fs, whereas the bleaching kinetics at 710 and 730 nm exhibited two components with time constants of 1 and 5.5 ps. Comparison of the kinetics of bleaching development at 710 nm and 730 nm with that of bleaching decay at 680 nm indicated that both long-wavelength forms of trimers are populated mainly via direct energy transfer from bulk chlorophyll.     

The effect of solvated ions on the thermal denaturation of human serum albumin in water-dimethylsulfoxide solutions

The effects of solvated ions on the thermal denaturation of human serum albumin (HAS) in water-dimethylsulfoxide (DMSO) solutions were studied by the method of electron absorption spectroscopy. It was shown that depending on the DMSO concentration, electrolytes (LiCl, LiNO₃, LiClO₄, NaCl, and NaNO₃) contained in these solutions were characterized by different anion and cation solvation degrees: unlike cations, anions were only negligibly solvated, which affected HAS thermal denaturation. Electrostatic interactions between anions and positively charged amino acid residues supporting protein denaturation subsided in the line Cl⁻ > NO₃⁻ > ClO₄⁻.     

Early stage aggregation of human serum albumin in the presence of metal ions

The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70 °C by monitoring the optical density changes at 400 nm on a 180 min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480 nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.     

Evidence of protein collective motions on the picosecond timescale

We investigate the presence of structural collective motions on a picosecond timescale for the heme protein, cytochrome c, as a function of oxidation and hydration, using terahertz (THz) time domain spectroscopy and molecular dynamics simulations. The THz response dramatically increases with oxidation, with the largest increase for lowest hydrations, and highest frequencies. For both oxidation states the THz response rapidly increases with hydration saturating above ∼25% (g H₂O/g protein). Quasiharmonic vibrational modes and dipole-dipole correlation functions were calculated from molecular dynamics trajectories. The collective mode density of states alone reproduces the measured hydration dependence, providing strong evidence of the existence of these motions. The large oxidation dependence is reproduced only by the dipole-dipole correlation function, indicating the contrast arises from diffusive motions consistent with structural changes occurring in the vicinity of buried internal water molecules. This source for the observed oxidation dependence is consistent with the lack of an oxidation dependence in nuclear resonant vibrational spectroscopy measurements.     

Determination of thermal properties of composting bulking materials

Thermal properties of compost bulking materials affect temperature and biodegradation during the composting process. Well determined thermal properties of compost feedstocks will therefore contribute to practical thermodynamic approaches. Thermal conductivity, thermal diffusivity, and volumetric heat capacity of 12 compost bulking materials were determined in this study. Thermal properties were determined at varying bulk densities (1, 1.3, 1.7, 2.5, and 5 times uncompacted bulk density), particle sizes (ground and bulk), and water contents (0, 20, 50, 80% of water holding capacity and saturated condition). For the water content at 80% of water holding capacity, saw dust, soil compost blend, beef manure, and turkey litter showed the highest thermal conductivity (K) and volumetric heat capacity (C) (K: 0.12–0.81 W/m °C and C: 1.36–4.08 MJ/m³ °C). Silage showed medium values at the same water content (K: 0.09–0.47 W/m °C and C: 0.93–3.09 MJ/m³ °C). Wheat straw, oat straw, soybean straw, cornstalks, alfalfa hay, and wood shavings produced the lowest K and C values (K: 0.03–0.30 W/m °C and C: 0.26–3.45 MJ/m³ °C). Thermal conductivity and volumetric heat capacity showed a linear relationship with moisture content and bulk density, while thermal diffusivity showed a nonlinear relationship. Since the water, air, and solid materials have their own specific thermal property values, thermal properties of compost bulking materials vary with the rate of those three components by changing water content, bulk density, and particle size. The degree of saturation was used to represent the interaction between volumes of water, air, and solids under the various combinations of moisture content, bulk density, and particle size. The first order regression models developed in this paper represent the relationship between degree of saturation and volumetric heat capacity (r = 0.95–0.99) and thermal conductivity (r = 0.84–0.99) well. Improved knowledge of the thermal properties of compost bulking materials can contribute to improved thermodynamic modeling and heat management of composting processes.     

Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below −40 °C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158–166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to −25 °C, holding them 10 min, rapidly cooling them to −70 °C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at −56 °C during warming and was complete by −46 °C. The present study differs in that the oocytes were warmed rapidly from −70 °C to temperatures between −65 and −50 °C and held for 3–60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (E_a) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 °C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 °C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 °C. These findings have significant implications both for cryobiology and cryo-electron microscopy.     

Protease production by a thermophilic Bacillus species (P-001A) which degrades various kinds of fibrous proteins

An aerobic Gram-positive sporeforming bacterium was isolated from an alkaline hot spring at Wondo Genet, Ethiopia. In an optimized culture medium it produced maximum activity of protease at 55°C and pH 9.5. The protease activity against casein was 65 units/ml. Enzyme activity was detected between 30–70°C and pH of 4.5–11.5. The enzyme had a half-life of 55 and 30 min at 60° and 70°C, respectively. The isolate hydrolysed 90, 60 and 50% of the skin, feather and horn used in the optimized medium within 120 h.