Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation

Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is synthesized in a prepro-form (prepro-Tk-subtilisin), secreted in a pro-form (pro-Tk-subtilisin), and matured to an active form (mat-Tk-subtilisin*; a Ca(2+)-bound active form of matured domain of Tk-subtilisin) upon autoprocessing and degradation of the propeptide [Tk-propeptide; propeptide of Tk-subtilisin (Gly1-Leu69)]. Pro-Tk-subtilisin exhibited halo-forming activity only at 80 degrees C, but not at 70 and 60 degrees C, because Tk-propeptide is not effectively degraded by mat-Tk-subtilisin* and forms an inactive complex with mat-Tk-subtilisin* at <80 degrees C. Random mutagenesis in the entire prepro-Tk-subtilisin gene, followed by screening for mutant proteins with halo-forming activity at 70 and 60 degrees C, allowed us to identify single Gly56 --> Ser mutation in the propeptide region responsible for low-temperature adaptation of pro-Tk-subtilisin. SDS-PAGE analyses and mat-Tk-subtilisin* activity assay of pro-G56S-subtilisin indicated more rapid maturation than pro-Tk-subtilisin. The resultant active form was indistinguishable from mat-Tk-subtilisin* in activity and stability, indicating that Gly56 --> Ser mutation does not seriously affect the folding of the mature domain. However, this mutation greatly destabilized the propeptide, making it unstructured in an isolated form. As a result, Tk-propeptide with Gly56 --> Ser mutation (G56S-propeptide) was more susceptible to proteolytic degradation and less effectively inhibited mat-Tk-subtilisin* activity than Tk-propeptide. These results suggest that pro-G56S-subtilisin is more effectively matured than pro-Tk-subtilisin at lower temperatures, because autoprocessed G56S-propeptide is unstructured upon dissociation from mat-Tk-subtilisin* and is therefore effectively degraded by mat-Tk-subtilisin*.     

Excellent properties of 0.15 micrometer ferroelectric PZT capacitor with SRO electrodes for future Gbit-scale ferams

Abstract

Excellent ferroelectric properties of PZT capacitors with 0.15μ m lateral dimension were obtained for the first time using SrRuO₃ electrode technology. The switching charge of the capacitor was not degraded even for the 0.15 μ m capacitor. The ferroelectric properties such as saturation, fatigue, and imprint characteristics were similar for the capacitors with 12 μ m to 0.15 μ m dimensions. We demonstrated that the SrRuO₃ electrode technology could be used for the future Gbit-scale FeRAMs. An apparent increase in switching charge was also observed with decrease in capacitor size. The ferro-film around the top electrode may contribute to the increase of switching charge.     

Importance of an extreme C-terminal motif of a family I.3 lipase for stability

A five-residue sequence motif (VTLVG) located at positions 15-19 from the C-terminus of family I.3 lipase from Pseudomonas sp. MIS38 (PML) and an extreme C-terminal motif (DGIVIA) located at the C-terminus of PML are relatively well conserved in the passenger proteins of type 1 secretion system (T1SS). To analyze the role of these motifs, four mutant proteins of PML (PMLΔ5, PMLΔ10, 3A-PML and 2A-PML) were constructed. PMLΔ5 and PMLΔ10 lack the C-terminal 5 and 10 residues of PML, respectively. 3A-PML has triple mutations within an extreme C-terminal motif and 2A-PML has double mutations within a five-residue sequence motif. Secretion of these proteins was analyzed using Escherichia coli DH5 cells carrying Lip system (T1SS for family I.3 lipase). The secretion level of 2A-PML was dramatically reduced when compared with that of PML, whereas the secretion level of 3A-PML was comparable to that of PML, indicating that a five-residue sequence motif, instead of an extreme C-terminal motif, is required for secretion of PML. None of the mutations and truncations seriously affects the enzymatic activity of PML. However, 3A-PML, PMLΔ5 and PMLΔ10 were less stable than PML by 2.1, 7.6 and 7.6°C in T(1/2), respectively, and by 5.0, 21.3 and 17.9 kJ/mol in ΔG(H(2)O), respectively. These results indicate that an extreme C-terminal motif of PML is important for stability.     

Development of method for estimating drop diameter in the manufacturing process of functional O/W microcapsules

A new method for preparing functional O/W microcapsules using a process involving O/W/O emulsion as particle formation was developed. Coenzyme Q10 (CoQ10) or reduced coenzyme Q10 (QH) was used as the core substance. QH oxidized fast when exposed to air. O/W microcapsules were manufactured by conventional liquid phase drying method (LPD). The purpose of this study is to develop a simple method of estimating drop diameter which is possible to evaluate immediately the mean drop diameter during the microencapsulation process without the usual photographic measurement. This developed estimation is possible to predict a Sauter mean diameter by measuring the amount of inner CoQ10 released from O/W emulsion droplet. The amount of inner oil phase released from O/W emulsion has correlation with increased total surface area of O/W emulsion droplet caused by breaking droplet. Released rate of CoQ10 from O/W emulsion droplet to outer continuous phase under different rotational speed and emulsion viscosity was measured with an absorption spectrometer. As a result of the changes of released inner CoQ10 amount, droplet breakage under low emulsion viscosity was promoted by agitation speed. It is concluded that droplet dispersion state during manufacturing of O/W microcapsules was evaluated well by applying the developed estimation method.     

The dynamic balance of import and export of zinc in Escherichia coli suggests a heterogeneous population response to stress

Zinc is essential for life, but toxic in excess. Thus all cells must control their internal zinc concentration. We used a systems approach, alternating rounds of experiments and models, to further elucidate the zinc control systems in Escherichia coli. We measured the response to zinc of the main specific zinc import and export systems in the wild-type, and a series of deletion mutant strains. We interpreted these data with a detailed mathematical model and Bayesian model fitting routines. There are three key findings: first, that alternate, non-inducible importers and exporters are important. Second, that an internal zinc reservoir is essential for maintaining the internal zinc concentration. Third, our data fitting led us to propose that the cells mount a heterogeneous response to zinc: some respond effectively, while others die or stop growing. In a further round of experiments, we demonstrated lower viable cell counts in the mutant strain tested exposed to excess zinc, consistent with this hypothesis. A stochastic model simulation demonstrated considerable fluctuations in the cellular levels of the ZntA exporter protein, reinforcing this proposal. We hypothesize that maintaining population heterogeneity could be a bet-hedging response allowing a population of cells to survive in varied and fluctuating environments.     

Phthoxazolin A, a specific inhibitor of cellulose biosynthesis from microbial origin. II. Isolation, physico-chemical properties, and structural elucidation

Phthoxazolin A is a new inhibitor of cellulose biosynthesis produces by Streptomyces sp. OM-5714. The active compound was isolated, and the structure was elucidated by spectrometric analyses.     

A method of noise removal using the inverse transform of an image autocorrelation function

A method for a reversible transform between an image and image autocorrelation function has been devised, and it has been applied to the reduction of random noise in a microscope image. Object-related features of an image signal as well as noise features are overlapped at the vicinity of the origin of an image autocorrelation, and a peak is formed at this position. The present method removes the noise contribution in this peak and returns the autocorrelation function, thus corrected, to the image. An autocorrelation function is computed by two Fourier transforms as based on the convolution theorem. The inverse transform from the image autocorrelation function is made possible by performing two Fourier transforms in reverse to the above. This method has been developed to remove random noise more accurately from a single image (especially for nonperiodic images) than other image-processing methods such as smoothing techniques and low pass filtering.     

Pneumatic fast-closing valve for synchrotron radiation beam linesat the photon factory

A fast-closing valve system has been designed and fabricated in order to protect the vacuum of the synchrotron radiation beam line and that of the 2.5 GeV storage ring from a sudden vacuum failure at the downstream end of the beam line. Upon the detection of a failure the valve closes within 11.9 ms by pneumatic pressure on a drive piston in a cylinder. Within the last 3.7 ms of the closing time the downward movement of the blade is decelerated so as to reduce the closing shock. A hard TiN ceramic membrane is coated on the surface of the titanium blade. On the surface of a stainless steel aperture, an Al₂O ₃-TiO₂ ceramic membrane (less hard than TiN) is coated in order to attain low conductance and smooth movement of the blade. This ensures a low leak rate of 0.135 torr l/s and a long life cycle of the mechanism (more than 8000 times)