The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the alpha-amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.
The effects of nonisobaric and isobaric steps on a two-bed O 2 pressure swing adsorption (PSA) packed with zeolite 5A were investigated. In addition, the operating conditions in the O 2 PSA were studied to supply the various O 2 concentrations to the oxygen aerator efficiently. Because the velocity variation along the bed and the related Mass Transfer Zone (MTZ) shape at the isobaric and nonisobaric steps were affected by the diffusion rate, the concentration-dependent rate parameter in a modified linear driving force (LDF) model was evaluated from the Darken equation related to the loading ratio correlation isotherm model. The LDF coefficients had a significant effect on the prediction of the O 2 purity and recovery at the low flow rate region. However, the higher the feed flow rate, the lower the LDF parameter effect. It was also noted that the effects of each step time in the isobaric and nonisobaric steps on the PSA performance were subtle in the O 2 purity and recovery except for the blowdown step. Also, there was the optimum step time to maximize the O 2 purity and recovery in the adsorption and pressurization steps, while the increase of O 2 purity and recovery with the pressurization equalization step time showed asymptotic curvature. The idle time at the pressurization step had a negative effect on the PSA performance, while the short idle time at the pressure equalization step led to improved product purity. However, it was negligible for the effect of the idle time at the blowdown step on the PSA performance. The successive variation between high and low P/F ratios caused the continuous variation of O 2 purity and recovery to nearly the same extent as the results under each constant P/F ratio condition. Therefore, to save the operating cost of the oxygen aerator system, the desired oxygen concentration for the aerator can be supplied simply by controlling the feed rate or purge rate.
Solubility measurements were performed in the temperature range of 298-333 K, for a methanol content varying between 0 mass % and 90 mass %. The phase equilibrium condition of L-ornithine-L-aspartate (LOLA) + water + methanol and the mean ionic activity coefficients of LOLA in a ternary system were calculated by using the Chen model, allowing the description of the mixed-solvent electrolyte systems. The mean ionic activity coefficients were calculated with nine binary parameters: six parameters for the binaries LOLA + water (taken from conductance measurements) and water + methanol (taken from the literature) and three parameters for the binary LOLA + methanol (obtained by regression of solubility data). Using binary adjustable parameters and solubility data, the model satisfactorily correlates the ternary (LOLA + water + methanol) system over the entire range of temperature and concentrations.
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