Soo Wan Nam

Fatigue perturbed creep of pure aluminum at ambient temperatures

The creep of 99.999 pct pure aluminum subject to tension-tension cyclic stressing (dynamic creep) has been studied and compared to creep for the peak cyclic stress applied statically (static creep). The creep rate for a cyclic stress of 29.8 to 3.7 MPa was always found to be greater than the creep rate for static application of a stress of 29.8 MPa over the temperature range of 302 K to 348 K. The ratio of the dynamic creep rate to the static creep rate was found to increase with increasing strain and decreasing temperature. The apparent activation energy measured for dynamic creep was less than that measured for static creep. The substructure for dynamic creep is softer than for static creep and probably accounts for the accelerated creep rate and the lower observed activation energy.

High-level secretion of human α1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence

The use of a proper signal sequence is one of the major determinants for the efficient secretion of heterologous proteins from yeast. The signal sequence derived from inulinase (INU1A) of Kluyveromyces marxianus was evaluated in directing the secretion of a human glycoprotein, alpha 1-antitrypsin (alpha 1-AT), from Saccharomyces cerevisiae. A yeast expression vector for alpha 1-AT was constructed by placing the coding sequence of human alpha 1-AT fused with the INU1A signal sequence downstream of the GAL10 promoter. S. cerevisiae transformants harboring the expression vector secreted about 70% of the total alpha 1-AT synthesized into the culture media. The intracellularly retained form of alpha 1-AT was mostly unglycosylated, whereas the secreted protein had high mannose-type glycosylation. The fed-batch cultivation of the recombinant yeast achieved a high-cell density, leading to the secretion of biologically active alpha 1-AT up to 75 mg l-1. The secreted protein was purified and subjected to N-terminal sequencing, which confirmed that the secreted alpha 1-AT was processed correctly at the Kex2 cleavage site as expected from the sequence of INU1A signal peptide. The results suggest that the inulinase signal sequence is useful for the high-level secretion of relatively large glycoproteins, such as human alpha 1-AT, from S. cerevisiae.

Highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides.

The INU genes of Kluyveromyces marxianus encode inulinases which are readily secreted from Saccharomyces cerevisiae into the culture medium. To evaluate the utility of the INU signal peptides for the secretion of heterologous proteins from S. cerevisiae, a variety of expression and secretion vectors were constructed with GAL10 promoter and GAL7 terminator. The coding sequence for human lipocortin-1 (LC1) was inserted in-frame with the INU signal sequences, and then the secretion efficiency and localization of LC1 were investigated in more detail and compared with those when being expressed by the vector with the MFalpha1 leader peptide. The vector systems with INU signal peptides secreted ca. 95% of the total LC1 expressed into the extracellular medium, while the MFalpha1 leader peptide-containing vector resulted in very low secretion efficiency below 10%. In addition, recombinant human interleukin-2 (IL-2) was expressed and secreted with the vector systems with INU signal peptide, and a majority fraction of the human IL-2 expressed was found to be secreted into the extracellular medium as observed in LC1 expression. (c) 1995 John Wiley & Sons, Inc.

New aspects on the superplasticity of fine-grained 7475 aluminum alloys

Thermomechanical processes were developed which give fine grain sizes of 6 and 8 μm in the 7475 Al alloy. Superplastic properties of this material were evaluated in the temperature range of 400 °C to 545 °C over the strain-rate range of 2.8 x 10-4 to 2.8 X 10-2 s-1. The maximum ductility exhibited by the alloy was approximately 2000 pct, and optimum superplasticity was achieved at a strain rate of 2.8 X 10-3 s-1 which is higher by an order of magnitude than other 7475 Al alloys. This result is attributed to the presence of fine dispersoids which maintain the fine grain size at high homologous temperatures. The flow stress and strain-rate sensitivity strongly depend on the grain size. The superplastic 7475 Al alloy has strain-rate sensitivities of 0.67 (6 μm) and 0.5 (13 μm) and an activation energy which is similar to the one for grain boundary diffusion of aluminum. Microstructural investigation after superplastic tests revealed zones free of dispersoid particles at grain boundaries primarily normal to the tensile direction. These dispersoidfree zones (DFZs) appear even after 100 pct elongation and are occasionally as large as 5 μm across. This result demonstrates the importance of diffusional flow in superplastic deformation of the fine-grained 7475 Al alloy especially at low elongations.

Continuous culture of a recombinantEscherichiacoli and plasmid maintenance for tryptophan production

SummaryProduction of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 ΔtrpLDtrpRtstna− (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.

Investigation of primary and secondary creep deformation mechanism of TiAl

Creep deformation behaviors in lamellar TiAl alloys have been investigated. As in the case with metals, the normal primary creep stage was observed. As creep strain increased within the primary regime, dislocation density decreased, and creep activation energy increased from 300 kJ/mol, the activation energy of the self-diffusion of Ti in TiAl, to about 380 kJ/mol, that of steady state creep deformation. During primary creep deformation of lamellar TiAl, as the initial dislocation density decreased, the α2 -phase was found to transform to a γ-phase, generating new dislocations which contributed to the creep deformation. In other words, this phase transformation is the source of the dislocation generation for continuous creep deformation. Therefore, we suggest that phase transformation is the rate controlling processes having an activation energy of about 400 kJ/mol, which is higher than that of self-diffusion. A small amount of prestrain was found to be responsible for the reduction of initial dislocation density. In addition, this prestrained specimen showed significantly reduced primary creep strain, and the creep activation energy in the primary stage was measured to be about 380 kJ/mol. These results clearly confirm the suggested creep deformation mechanism of lamellar TiAl alloys.

Characteristics of dislocation structure in creep deformed lamellar tial alloy within primary regime

In this investigation, dislocations of a lamellar TiAl alloy are analyzed after creeping in the primary range at 800°C/200MPa in order to interpret their mobility It was found that the dislocation density in γ-laths decreased as the creep deformation proceeds within primary creep regime Schmid factor analysis suggests that the creep deformation in the early stage of the primary creep regime is controlled by the gliding of some of the initial dislocations which have a high enough Schmid factor As the creep deformation progressed, those dislocations with high Schmid factors slip preferentially to be annihilated at the α-γ interface For further continuous deformation, dislocation generation is required, and for this, α-phase is transformed to γ-phase in order to generate new dislocations A slow dislocation generation process by phase transformation of α-phase compared with the absorbing rate to sinks is responsible for the decreasing dislocation density as the creep strain increases

The use of inulinase pre-pro leader peptide for secretion of heterologous proteins in Saccharomyces cerevisiae

SummaryThe pre-pro and pre regions of inulinase leader peptide have been used to secrete the heterologous proteins such as human lipocortin-1 and human interleukin-2 (IL2) in Saccharomyces cerevisiae. The inulinase leader peptide consisting of both pre and pro regions directed an efficient secretion of such heterologous proteins in S. cerevisiae, while deletion of pro region prevents them from being secreted. Both pre and pro regions of inulinase leader peptide appears to be necessary to direct secretion of heterologous proteins in S. cerevisiae.

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

Abstract On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O2, CO2, H2O, and N2 contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.

Rapid purification of recombinant human lipocortin-I secreted fromSaccharomyces cerevisiae

Human lipocortin-I was expressed as a secretory product bySaccharomyces cerevisiae harboring an expression system consisting ofGAL10 promoter, inulinase signal sequence and lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys26.