Increased proteolytic resistance of ribonuclease A by protein engineering.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.

Preparation of calibration materials for microanalysis of Ti minerals by direct fusion of synthetic and natural materials: experience with LA-ICP-MS analysis of some important minor and trace elements in ilmenite and rutile.

Calibration materials for microanalysis of Ti minerals have been prepared by direct fusion of synthetic and natural materials by resistance heating in high-purity graphite electrodes. Synthetic materials were FeTiO3 and TiO2 reagents doped with minor and trace elements; CRMs for ilmenite, rutile, and a Ti-rich magnetite were used as natural materials. Problems occurred during fusion of Fe2O3-rich materials, because at atmospheric pressure Fe2O3 decomposes into Fe3O4 and O2 at 1462 degrees C. An alternative fusion technique under pressure was tested, but the resulting materials were characterized by extensive segregation and development of separate phases. Fe2O3-rich materials were therefore fused below this temperature, resulting in a form of sintering, without conversion of the materials into amorphous glasses. The fused materials were studied by optical microscopy and EPMA, and tested as calibration materials by inductively coupled plasma mass spectrometry, equipped with laser ablation for sample introduction (LA-ICP-MS). It was demonstrated that calibration curves based on materials of rutile composition, within normal analytical uncertainty, generally coincide with calibration curves based on materials of ilmenite composition. It is, therefore, concluded that LA-ICP-MS analysis of Ti minerals can with advantage be based exclusively on calibration materials prepared for rutile, thereby avoiding the special fusion problems related to oxide mixtures of ilmenite composition. It is documented that sintered materials were in good overall agreement with homogeneous glass materials, an observation that indicates that in other situations also sintered mineral concentrates might be a useful alternative for instrument calibration, e.g. as alternative to pressed powders.

Differentiation between conformational and autoproteolytic stability of the neutral protease from Bacillus stearothermophilus containing an engineered disulfide bond.

The introduction of a disulfide bond into the neutral protease from Bacillus stearothermophilus by the double mutation G8C/N60C had resulted in an extremely thermostable enzyme with a half-life of 35.9 min at 92.5 degrees C [Mansfeld, J., Vriend, G., Dijkstra, B.W., Veltman, O.R., van den Burg, B., Venema, G., Ulbrich-Hofmann, R. & Eijsink, V.G. (1997) J. Biol. Chem. 272, 11152-11156]. The study in guanidine hydrochloride of this enzyme and the respective wild-type enzyme allowed us to distinguish between the stability toward global unfolding and autoproteolysis. At low protease concentrations (20 microg.mL-1) and short periods of incubation with guanidine hydrochloride (5 min), transition curves without the interference by autoproteolysis could be derived from fluorescence emission measurements. The effect of the disulfide bond on the global unfolding of the protein proved to be smaller than expected. In contrast, the measurement of autoproteolysis at higher protein concentrations (100 microg.mL-1) by quantitative evaluation of the bands of intact protein on SDS/PAGE revealed a strong stabilization toward autoproteolytic degradation by the disulfide bond. The rate of autoproteolysis in guanidine hydrochloride was found to be much lower than that of thermal denaturation, which can be attributed to the inhibition of the proteases by this denaturant. The results suggest that the disulfide bond stabilizes the protease against autoproteolysis more than against global unfolding. Autoproteolysis starts as soon as the cleavage sites in flexible external structural regions become accessible. It is suggested that the stabilizing effect of the disulfide bond is caused by the fixation of the crucial loop region 56-69 or by hindrance of the primary cleavage in this region by the amino acid exchanges.

Site-specific and random immobilization of thermolysin-like proteases reflected in the thermal inactivation kinetics.

Immobilization of proteins usually leads to random orientation of the molecules on the surface of the carrier material, whereby mechanistic interpretations of changes in properties, such as thermal stability, become very difficult. Recently, we have prepared several mutant enzymes of the thermolysin-like neutral protease from Bacillus stearothermophilus, containing cysteine residues in different positions on the surface of the protein molecule. These enzymes allowed site-specific immobilization to Activated Thiol-Sepharose and showed that stabilization effects strongly depend on the position of attachment [Mansfeld, Vriend, Van den Burg, Eijsink and Ulbrich-Hofmann (1999) Biochemistry 38, 8240-8245]. The greatest stabilization was achieved after immobilization of the mutant enzymes S65C and T56C/S65C within the structural region (positions 56-69) where unfolding is initiated. In this study thermal inactivation kinetics of these two mutant enzymes, as well as those of the pseudo-wild-type enzyme and thermolysin, were compared for different types of immobilization. Besides site-specific immobilization via thiol groups, the enzymes were bound randomly via their amino groups or by mixed-type binding. The basic matrix was Sepharose 4B in all carriers. Whereas the enzymes bound site-specifically to Activated Thiol-Sepharose showed clear first-order inactivation kinetics like the soluble enzymes, the other immobilized enzyme preparations were characterized by distinct biphasic inactivation kinetics reflecting the heterogeneity of enzyme molecules on the carrier with respect to thermal unfolding. Site-specific binding resulted in stronger stabilization than the mixed binding type. However, immobilization to a highly functionalized carrier via amino groups increased stability further, suggesting that multiple fixation outside of the unfolding region 56-65 is able to increase stability of the enzyme molecules additionally.

Characterization of a novel stable biocatalyst obtained by protein engineering.

Protein engineering is a powerful tool for the improvement of the properties of biocatalysts. Previously we have applied protein engineering technologies to obtain an extremely stable variant of the thermolysin-like protease from Bacillus stearothermophilus [Van den Burg, Vriend, Veltman, Venema and Eijsink (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2056-2060]. This variant is much more resistant to denaturing conditions (temperature and denaturing agents) than the wild-type enzyme. An extensive enzymic characterization was undertaken to explore the suitability of the variant as a biocatalyst at high temperatures. By comparing a range of variants with increasing thermal stabilities we show that the additivity of the mutations is accompanied by an increase in activity at elevated temperatures in accordance with the Arrhenius law. The results suggest that the constructed protease variants could be suitable alternatives to proteases that are currently used industrially. Our studies demonstrate how protein engineering can be exploited to obtain high-performance biocatalysts.

Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

Mutational analysis of a surface area that is critical for the thermal stability of thermolysin-like proteases.

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55-69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57-->Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.

Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond.

The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

Application of polystyrene-bound invertase to continuous sucrose hydrolysis on pilot scale.

Invertase from baker's yeast (Saccharomyces cerevisiae) covalently bound to a macroporous polystyrene anion-exchange resin via glutaraldehyde was applied to continuous sucrose hydrolysis in packed bed-reactors. The process was scaled up from 3-mL laboratory reactors via 0.3-L reactors to pilot-scale 50-L reactors without significant loss of efficiency. The described process allows the production of a wide spectrum of invert sugar syrups with high purity in continuous procedure. The 50-L reactor was used under process conditions 1 year without significant loss of productivity at a temperature of 40 degrees C. A productivity of 760 g/h was obtained with 1 L invertase-polystyrene complex using a 2.5M sucrose solution as substrate.

A prospective trial using salivary-theophylline levels to guide asthma therapy.

In three prospectively designed studies, first saliva (SA) samples were collected simultaneously with serum (SE) from 86 children who received slow-release theophylline (Th) b.i.d. for treatment of bronchial asthma. SA was collected with a standardized sampling procedure. Regression analysis revealed a close correlation between SE and SA-Th levels (r = 0.959; SE = 1.87 x SA + 0.05) and yielded a "therapeutic SA-Th range" of 5.8 to 10.7 mg/L. In 16 children the diurnal variation was then assessed by measuring 6 SA-Th levels within 24 hr, using the same sampling method. At 0, 9, and 24 hr SE-Th levels were also measured. The levels of SE-Th and SA-Th were again closely correlated (0.914 less than or equal to r less than or equal to 0.949) and both remained constant over the 24-hr period. In the third prospective trial only SA levels were used to monitor Th therapy in 50 asthmatic children. Seven days after starting therapy SA-Th levels were measured and then the Th dose was increased weekly until SA-Th levels above 5.8 mg/L were reached. The following day SE and SA samples were collected simultaneously and their Th levels compared. In 36 of the 47 children who completed the study therapeutic SE-Th levels (greater than or equal to 10 and less than or equal to 20 mg/L) were measured; in one patient it was greater than or equal to 20 mg/L, in 6 it was between 8.0 and 10 mg/L, and only 4 had SE levels less than 8.0 mg/L.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of invertase with polyelectrolytes.

In connection with our work on polyelectrolyte complex formation with polyampholytes, the interaction between invertase and several linear polyelectorlytes has been investigated by means of turbidimetry, light scattering measurements, and determination of the enzyme activity. Polyelectrolyte complex formation of invertase was shown to occur with cationic polyelectrolytes only. The light-scattering data yield information on aggregation and desegregation processes in complex formation. As indicated by our results, only a part of the protein molecules is engaged in this Coulombic interaction, and this part shows a rather small enzyme activity only. Thus, a direct interaction between invertase and a cationic polyelectrolyte is no effective approach to enzyme binding, but a complete immobilization of invertase can be achieved via an "inclusion flocculation" with a symplex formed by interaction between an anionic and a cationic linear polyelectrolyte or via immobilization in symplex microcapsules.

Immobilization of invertase by encapsulation in polyelectrolyte complexes.

Free and polystyrene-bound invertase from Saccharomyces cerevisiae were encapsulated within symplex membranes which were composed of cellulose sulfate as the polymeric anion and poly(dimethyldiallylammonium chloride) as the polymeric cation. The kinetics and the performance of the encapsulated enzyme preparations have been compared to the free enzyme employing the hydrolysis of sucrose. The pH and temperature optima were only slightly affected by the encapsulation. The kinetic constants, however, were changed by the encapsulation as a result of diffusional limitation. Encapsulated invertase showed a high storage stability and a high operational stability if low substrate concentrations were applied. The coimmobilization of invertase with living cells, which are not capable of utilizing sucrose, in the described capsules, opens many possibilities in fermentation technology.

[Comparison of salivary with serum levels of retard theophylline under steady-state conditions]. / Vergleich von Saliva- mit Serumspiegeln bei Retardtheophyllin unter Steady-State-Bedingungen.

In 86 asthmatic children, the theophylline levels in the serum and saliva were compared. A pronounced (r = 0.95) and statistically highly significant (p less than 0.001) relationship was observed to exist between the theophylline concentrations in serum and saliva.

Invertase immobilized on macroporous polystyrene: properties and kinetic characterization.

Invertase from baker's yeast (Saccharomyces cerevisiae) was covalently bound via benzoquinone and glutaraldehyde to a macroporous polystyrene anion exchanger. The behavior of the invertase-polystyrene complexes in batch and packed-bed reactors was characterized kinetically. In addition to kinetic studies on sucrose hydrolysis at low initial substrate concentrations, the dependence of conversion degree on flow rate at high, industrially used substrate concentrations was determined. The described invertase-polystyrene complexes are suitable for technical application in the production of glucose-fructose mixtures because of their high specific and relative activities, as well as the good hydrodynamical and mechanical properties of the polystyrene matrix.

[Synthesis of cyclic and cyclically branched tachykinin partial sequences. 1. Synthesis of the homodet-cyclic eledoisin(6-11)-hexapeptide]. / Synthese cyclischer und cyclisch-verzweigter Tachykinin-Teilsequenzen. Teil 1: Darstellung des homodet-cyclischen Eledoisin(6-11)-Hexapeptides.

Ala-Phe-Ile-Gly-Leu-Met has been disengaged by cyclization of H-Leu-Met-Ala-Phe-Ile-Gly-OH by means of dicyclohexylcarbodiimide/N-Hydroxysuccinimide or the adequate p-nitrophenylester. Acc. to various strategic variants, the design of the linear precursor has been performed by condensation of the segments of Boc-Leu-Met-OH and H-Ala-Phe-Ile-Gly-OH. The resulting cyclo-[Eledoisin(6-11)-Hexapeptide] has in a clearly separated range of dose dual agonistic and antagonistic effects at the guinea-pig ileum.