A highly-active endo-1,3-1,4-β-glucanase from thermophilic Talaromyces emersonii CBS394.64 with application potential in the brewing and feed industries

A 1245-bp endoglucanase gene of glycoside hydrolase (GH) family 7, egl7A, was cloned from the acidothermophilic fungus Talaromyces emersonii CBS394.64 and successfully expressed in Pichia pastoris. Sequence alignments indicated that Egl7A had highest identity of 62.7% at the amino acid level with the functionally characterized endoglucanase from Aspergillus terreus NIH2624. Purified recombinant Egl7A exhibited the maximum activity at pH 4.5 and 70 °C, retained stable over the pH range of 2.0–12.0 and at 65 °C, and was strongly resistant to acidic and neutral proteases, most metal ions and SDS. The enzyme exhibited the highest specific activity reported so far (11,299 U mg⁻¹) when using barley β-glucan as the substrate. Egl7A exhibited broad substrate specificity, including barley β-glucan, lichenin, CMC-Na, and xylan and had capacity to cleave cellopentaose and cellohexaose into smaller units rapidly. Under simulated mashing conditions, addition of Egl7A reduced the mash viscosity by 12.40%; when combined with a GH10 xylanase, more viscosity reduction (27.75%) was observed, which is significantly higher than that of the commercial enzyme Ultraflo XL (17.91%). All these properties make Egl7A attractive for potential applications in the feed and brewing industries.     

Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated β-glucosidase from the thermophilic fungus Humicola insolens

A mycelial β-glucosidase from the thermophilic mold Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94 kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55 kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Humicola grisea var. thermoidea, with about 22% coverage. Optima of temperature and pH were 60 °C and 6.0–6.5, respectively. The enzyme was stable up to 1 h at 50 °C and showed a half-life of approximately 44 min at 55 °C. The β-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-fucopyranoside, p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-galactopyranoside, o-nitrophenyl-β-d-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-β-d-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400 mM, with maximal stimulatory effect (about 2-fold) around 40 mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials.     

Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Laboratory evolution of laccase for substrate specificity

A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity, and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild-type CotA oxidizes ABTS (ABTS = diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and SGZ (SGZ = 4-hydroxy-3,5-dimethoxy-benzaldehyde azine), and it was engineered for increased specificity for ABTS. Based on the ABTS-bound crystal structure of CotA, 19 amino acids are within 6 Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half-life for the heat inactivation (t_1/2) at 80 °C was increased by 62 min for the mutant. Laccases have several applications in biotechnology, which include pulp bleaching, biosensors, bioremediation, and biofuel cells. The substrate specificity of CotA is moldable and the enzyme can be tailored to oxidize a variety of target molecules for specific purposes.     

Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation

Covalent attachment of surface proteins to the cell wall of Gram-positive bacteria requires a sortase-mediated transpeptidation reaction. In almost all Gram-positive bacteria, the housekeeping sortase, sortase A, recognizes the canonical recognition sequence LPXTG (X = any amino acid). The human pathogen Clostridium difficile carries a single putative sortase gene (cd2718) but neither transpeptidation activity nor specificity of CD2718 has been investigated. We produced recombinant CD2718 and examined its transpeptidation activity in vitro using synthetic peptides and MALDI-ToF(-ToF) MS analysis. We demonstrate that CD2718 has sortase activity with specificity for a (S/P)PXTG motif and can accommodate diaminopimelic acid as a substrate for transpeptidation.     

Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361

The gene encoding a family 5 endoglucanase, cel5A, was cloned from the moderate thermophile Bacillus licheniformis strain B-41361. The primary structure of the translated cel5A gene predicts a 49 amino acid putative secretion signal and a 485 residue endoglucanase consisting of an N-terminal family 5 catalytic domain and C-terminal family 3 cellulose binding domain. The endoglucanase portion of the gene was expressed in Escherichia coli, but soluble activity in cell lysates was due to a truncated enzyme with an apparent mass of 42 kDa, the equivalent of the predicted catalytic domain. Insoluble protein renatured from inclusion bodies was protected against truncation, yielding an active holoenzyme (rCel5A) with apparent mass of 62 kDa. The recombinant rCel5A was optimally active at 65 °C and pH 6.0, but retained only 10% activity after 1 h incubation at this temperature. At 55 °C, rCel5A had a broad pH range for activity and stability, with greater than 75% relative activity from pH 4.5–7.0, and retaining greater than 80% relativity activity across the range pH 4.5–8.0 following 1 h incubation at 55 °C. It readily hydrolyzed pNPC, carboxymethylcellulose, barley β-glucan, and lichenan, but despite binding to cellulose, had only weak activity against avicel. Hydrolysis products from soluble polysaccharides included glucose, cellobiose, cellotriose, and cellotetraose. The catalytic properties, broad pH range and thermostability of the recombinant B. licheniformis endoglucanase may prove suitable for industrial applications.     

Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster

The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7 Å resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a β-sheet and three α-helices forming a cone-like shape. However, known binding sites for RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site.     

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73 U/mg) at pH5 and 50 °C. The K_m and V_max with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20 mM and 2.41 U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140 mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.     

Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere

An extracellular lipase (EC 3.1.1.3), SAL-PP1, from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere was purified and characterized. The enzyme was purified using PALL'S Microsep centrifugal device (10 kD cut off), hydrophobic interaction (phenyl sepharose CL-4B column) and Superose-12 gel filtration chromatography and found to have a molecular mass of around 49 kDa. The gene fragment encoding the part of the catalytic site of the SAL-PP1 lipase was sequenced and the deduced amino acid sequence shows 93% identity with that of SEL3. SAL-PP1 showed activity against long acyl-chain triglycerides, various p-nitrophenyl esters and phospholipids. The enzyme shows high stability and activity after incubation with various metal ions (retained >90% activity in presence of Ca²⁺, Na⁺, Cu²⁺, Mg²⁺, Fe²⁺, or Hg²⁺ at 10 mM), organic solvents (retained >80% activity in presence of acetonitrile, ethanol, DMSO, methanol, isopropanol, toluene, or ethylene glycol at 10 mM), detergents (retained >70% activity in Triton X-100, Tween 80, or sodium deoxycholate at 10 mM) and irreversible inhibitors (retained >77% activity in presence of PMSF, leupetin, or β-mercaptoethanol, at 1 mM). Thermal inactivation studies revealed a temperature dependent unfolding of secondary structure of protein. SAL-PP1 showed maximal activity and stability at pH 8.0 and pH 9.0, respectively. The alkali-thermostability, organic solvent-tolerance and broad substrate specificity of this enzyme may have potential implications in detergent formulations, biotransformation, industries, and medicine.     

Crystal structure of cce_0566 from Cyanothece 51142, a protein associated with nitrogen fixation in the DUF269 family

The crystal structure for cce_0566 (171 aa, 19.4 kDa), a DUF269 annotated protein from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142, was determined to 1.60 Å resolution. Cce_0566 is a homodimer with each molecule composed of eight α-helices folded on one side of a three strand anti-parallel β-sheet. Hydrophobic interactions between the side chains of largely conserved residues on the surface of each β-sheet hold the dimer together. The fold observed for cce_0566 may be unique to proteins in the DUF269 family, hence, the protein may also have a function unique to nitrogen fixation. A solvent accessible cleft containing conserved charged residues near the dimer interface could represent the active site or ligand-binding surface for the protein’s biological function.Structured summary of protein interactionsDUF269 and DUF269 bind by x-ray crystallography (View interaction)     

A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110 kDa has an optimal pH of 7.0 and a pI of 5.6. It splits β-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl β-naphthylamide (Leu βNA) is the best substrate with the highest hydrolytic coefficiency followed by Met βNA = Arg βNA = Lys βNA > Ala βNA > Tyr βNA > Phe βNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K_m 82 μM and a k_cat of 1.08 s⁻¹, and Met-enkephalin with a K_m of 106 μM and a k_cat of 2.6 s⁻¹. The puromycin-sensitive enzyme is most susceptible to amastatin with an IC₅₀ of 0.05 μM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

Isolation,structure and biological activity of phomafungin,a cyclic lipodepsipeptide from a widespread tropical Phoma sp.

We isolated a cyclic lipodepsipeptide, phomafungin, from a Phoma sp. The distinct antifungal activity of phomafungin in the crude extract was initially discovered by mechanistic profiling in the Candida albicans fitness test. The purified compound contains a 28 member ring consisting of eight amino acids and a β-hydroxy-γ-methyl-hexadecanoic acid, and displays a broad spectrum of antifungal activity against Candida spp., Aspergillus fumigatus and Trichophyton mentagrophytes with MIC of 2–8 μg/ml, and toxicity to mice at 25 mg/kg. The linear peptide derived from opening of the lactone ring was devoid of antifungal activity as well as toxicity. Phomafungin has been identified in a number of Phoma spp. collected from Africa and the Indian and Pacific Ocean islands.     

Molecular and biochemical characterizations of a novel arthropod endo-β-1,3-glucanase from the Antarctic springtail,Cryptopygus antarcticus,horizontally acquired from bacteria

Collembolan species have been known to have β-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-β-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813 bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E–I–D–I–T–E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (K_m ～ 9.98 mg/mL) with an optimal temperature 50 °C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-β-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial β-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63 bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.     

A novel thermophilic xylanase from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing and other industries

Thermophilic xylanases are of great interest for their wide industrial application prospects. Here we identified a thermophilic xylanase (XynC01) of glycoside hydrolase (GH) family 10 in a thermophilic fungal strain Achaetomium sp. Xz-8. The deduced amino acids of XynC01 showed the highest identity of ≤52% to experimentally verified xylanases. XynC01 was functionally expressed in Pichia pastoris, showed optimal activity at pH 5.5 and 75 °C with stability over a broad pH range (pH 4.0–10.0) and at temperatures of 55 °C and below. XynC01 had the highest catalytic efficiency (k_cat/K_m, 3710 mL/s/mg) ever reported for all GH 10 xylanases, and was resistant to all tested metal ions and chemical reagents. Its hydrolysis products of various xylans were simple, mainly consisting of xylobiose and xylose. Under simulated mashing conditions, XynC01 alone had a comparable effect on filtration improvement with Ultraflo from Novozymes (20.24% vs. 20.71%), and showed better performance when combined with a commercial β-glucanase (38.50%). Combining all excellent properties described above, XynC01 may find diverse applications in industrial fields, especially in the brewing industry.     

Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461

An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36 kDa. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (v_max), apparent Michaelis–Menten constant (K_m) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (k_cat) were calculated as 7.63 U mg⁻¹, 0.204 mM, 4.42 mM and 697.4 min⁻¹, respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates.     

The in vivo assessment of mechanical loadings on pectoral pacemaker implants

Reduced sizes of implantable cardiac pacemakers and clinical advances have led to a higher feasibility of using such devices in younger patients including children. Increased structural demands deriving from reduced device size and more active recipients require detailed knowledge of in vivo mechanical conditions to ensure device reliability. Objective of this study was the proof of feasibility of a system for the measurement of in vivo mechanical loadings on pacemaker implants. The system comprised the following: implantable instrumented pacemaker (IPM) with six force sensors, accelerometer and radio-frequency (RF) transceiver; RF data logging system and video capture system. Three Chacma baboons (20.6±1.15 kg) received one pectoral sub-muscular IPM implant. After wound healing, forces were measured during physical activities. Forces during range of motion of the arm were assessed on the anaesthetized animals prior to device explantation. Mass, volume and dimensions of the excised Pectoralis major muscles were determined after device explantation. Remote IPM activation and data acquisition were reliable in the indoor cage environment with transceiver distances of up to 3 m. Sampling rates of up to 1000 Hz proved sufficient to capture dynamic in vivo loadings. Compressive forces on the IPM in conscious animals reached a maximum of 77.2±54.6 N during physical activity and were 22.2±7.3 N at rest, compared with 34.6±15.7 N maximum during range of motion and 13.4±3.3 N at rest in anaesthetized animals. The study demonstrated the feasibility of the developed system for the assessment of in vivo mechanical loading conditions of implantable pacemakers with potential for use for other implantable therapeutic devices.