Production of recombinant human lysosomal acid lipase in Schizosaccharomyces pombe: development of a fed-batch fermentation and purification process

A fed-batch fermentation process has been developed to enable the production of large quantities of recombinant human lysosomal acid lipase (hLAL; EC 3.1.1.13), in Schizosaccharomyces pombe, for preclinical studies as a potential enzyme therapy drug. Recombinant S. pombe, clone ASP397-21, expressed enzymatically active hLAL in the secreted form. A feedback fed-batch system was used to determine the optimal feed rate of a 50% glucose solution used as the carbon source. The feed rate of the glucose solution was calculated by a computer-aided system according to the equation; F=q(sf)(VX)/S(in) (q(sf), specific substrate feed rate [gram substrate/gram dry cell weight/h]; V, volume of culture broth [l]; X, cell density [gram dry cell weight/l]; S(in), concentration of growth limiting substrate in feed solution [gram substrate/gram feed solution]). At the time of the initial consumption of glucose in the batch-phase culture, the nutrient supply was automatically initiated by means of monitoring the respiratory quotient change. The obtained profile of the feed rate was applied to the feed forward control fermentation. Finally, the cells were grown up to >50 g dry cell weight/l, and the hLAL expression level was approximately 16,000 U/l. Expressed hLAL protein was purified in a two-step process by hydrophobic interaction and anion exchange chromatographies. Purified recombinant hLAL exhibited a 90-150 kDa broad band upon SDS-PAGE with specific activity of about 300 U/mg. After endoglycosidase H treatment, the band converged to 45 kDa, equal to the calculated molecular weight, suggesting that hLAL produced in S. pombe was hyper-glycosylated. N-terminal analysis of de-glycosylated hLAL revealed that the signal sequence of hLAL was correctly processed in S. pombe.     

Devolution, new regionalism and economic revitalization in Japan: Emerging urban political economy and politics of scale in Osaka-Kansai

Determining that the centralized political system is the cause of its persistent economic problems, Japan’s political leaders have been promoting neoliberalism-oriented state decentralization since the early 2000s. Stimulated by this policy, the prefecture of Osaka - the center of Japan’s second economic region - and its neighbors established a federation-type regional government in December 2010. However, some members left this regionalist coalition before its launch, which demonstrates the existance of internal tensions. In this study, the frameworks of Institutional Collective Action and politics of scale are applied to examine the regionalism decision-making process. Our findings suggest: (1) systemic political and economic biases run within the regionalism and (2) the likely urban economic and governance conditions after the devolution would be much more uncertain and messier than the reform advocates suggest they would be.     

Ultrastructural analysis of buckwheat starch components using atomic force microscopy

Morphological and structural features of buckwheat starch granules and nanocrystals were examined using atomic force microscopy and dynamic light scattering. Partially digested starch granules revealed a clear pattern of growth rings with the central core revealing lamellar structure. Atomic force microscopy and dynamic light scattering experiments revealed that the buckwheat starch granules were polygonal in shape and were in the range of 2 to 19 μm in diameter. The optimized acid hydrolysis process produced nanocrystals with the shape of spherical structure with lengths ranging from 120 to 200 nm, and the diameter from 4 to 30 nm from aqueous suspensions of buckwheat starch solution. The sorption isotherms on buckwheat starch nanocrystal/glycerol composite exhibited a 3-stage transition of moisture in the blending. The biocompatible nature of buckwheat starch nanocrystals and their structural properties make them a promising green nanocomposite material. PRACTICAL APPLICATION: Buckwheat starches had never been studied on a nanoscale, but we have achieved new understanding of starch granule morphology and concentric growth rings using nanoscale imaging. Since buckwheat is an underutilized crop, we foresee the potential application of buckwheat starch, starch-based nanocrystals, and nanoparticles, to expand markets and encourage producers to expand their buckwheat acreage. The atomic force image analysis suggests that buckwheat starch could be used as a new biopolymer material in food industries.     

Waste from Biodiesel Manufacturing as an Inexpensive Carbon Source for Bioreactors Treating Acid Mine Drainage

Abstract.  Alcohol-fed, semi-passive bioreactors have been used to support the growth of sulfate-reducing bacteria (SRB) for treatment of acid drainage from mine sites. An alcohol source not previously examined for use in these reactors is the glycerol-methanol waste remaining after the production of biodiesel fuel. In the laboratory, rock-filled columns were used to investigate biodiesel waste (BDW) as a carbon source for SRB. Columns were provided with water containing 900 mg/L sulfate, and fed reagent-grade glycerol or BDW in sufficient quantity to reduce 50% of the sulfate. Addition of 246 mg/L of reagent-grade glycerol resulted in 50% sulfate reduction and production of up to 59 mg/L of soluble sulfide, while the equivalent of 246 mg/L of glycerol provided as BDW resulted in 55% sulfate reduction and the production of up to 92 mg/L of soluble sulfide. During the initial stages of acclimation, propionic, acetic, formic, and lactic acids were observed. Acid concentrations were reduced over time in the effluent, and organic carbon in the BDW was nearly completely converted to carbon dioxide.     

Evaluation of Milk Quality in Delivering Sterilized Milk with Soft Tank Transportation System

ABSTRACT:  A new transportation system is proposed recently to improve the defects of liquid transportation by tank trucks. This method is called "soft tank transportation system"; a driver installs a sac-like container (soft tank), which is made from a tarpaulin with high-pressure resistant-waterproof zippers, in a general cargo vehicle. To evaluate the quality of sterilized milk by using the soft tank transportation system, ground and marine transportation for a long distance which took about 36 h from the shipper's loading to the receiver's unloading in a high-temperature summer season (average outside temperature was 33.4 °C) were carried out. Although the difference of milk temperature before and after the delivery varied from −0.7 to +1.4 °C, there was no difference in milk quality (fat, nonfat solids, total dissolved solids, and pH) and no coliform bacteria were detected. It can be evaluated that sterilized milk was carried in keeping good conditions by soft tank transportation system.     

Remote laser welding applications for car bodies

The fundamental objective of this study is to ensure the safety of nuclear reactors. A few accidents involving leaks from welded zones at the pipe penetration part of reactor vessels or at coolant pipes have been reported at home and abroad. One of the main causes is welding residual stress. Therefore, it is very important to know the welding residual stress in order to maintain the high safety of the plant, estimate the plant life cycle and design an effective maintenance plan. Welded joints of nuclear reactor vessels have complex shapes, and the welding residual stresses also have three-dimensional (3D) complex distributions. In this study, inherent strain-based theory and method are applied to measure the welding residual stresses. The inherent strain method is an analytical method as an inverse problem, using the least squares method, based on the finite element method. So the method gives the most probable value and deviation of residual stress. The reliability of the estimated result is discussed. In this method, inherent strains are unknowns. When residual stresses are distributed complexly in a 3D stress-state, the number of unknowns becomes very large. So, the inherent strain distribution is expressed with an appropriate function to decrease largely the number. A mock-up is idealized for a welded joint at the pipe penetration part of an actual reactor vessel. The inherent strain method is applied to the measure the residual stress of the joint. In this paper, the applicability of the inherent strain distribution function is diagnosed. Ten kinds of functions are applied to estimate the residual stress, and the accuracy and reliability of the analysed results are judged from three points of view, i.e. residuals, unbiased estimate of variance of errors and welding mechanics. The most suitable function is selected, which brings the most reliable result.     

An Extremely Stable,Highly Soluble Monosubstituted Anthraquinone for Aqueous Redox Flow Batteries

An extremely stable, energy-dense (53.6 Ah L⁻¹, 2 m transferrable electrons), low crossover (permeability of <1 × 10⁻¹³ cm² s⁻¹ using Nafion 212 (Nafion is a trademark polymer from DuPont)), and potentially inexpensive anthraquinone with 2-2-propionate ether anthraquinone structure (abbreviated 2-2PEAQ) is synthesized and extensively evaluated under practically relevant conditions for use in the negolyte of an aqueous redox flow battery. 2-2PEAQ shows a high stability with a fade rate of 0.03–0.05% per day at different applied current densities, cut-off voltage windows, and concentrations (0.1 and 1.0 m ) in both a full cell paired with a ferro/ferricyanide posolyte as well as a symmetric cell. 2-2PEAQ is further shown to have extreme long-term stability, losing only ≈0.01% per day when an electrochemical rejuvenation strategy is employed. From post-mortem analysis (nuclear magnetic resonance (NMR), liquid chromatography–mass spectrometry (LC-MS), and cyclic voltammetry (CV)) two degradation mechanisms are deduced: side chain loss and anthrone formation. 2-2PEAQ with the ether linkages attached on carbons non-adjacent to the central ring is found to have three times lower fade rate compared to its isomer with ether linkages on the carbon adjacent to the central quinone ring. The present study introduces a viable negolyte candidate for grid-scale aqueous organic redox flow batteries.