Characterization of Acetylxylan Esterase from White-Rot Fungus Irpex lacteus

The carbohydrate esterase family 1 (CE1) in CAZy contains acetylxylan esterases (AXEs) and feruloyl esterases (FAEs). Here we cloned a gene coding for an AXE belonging to CE1 from Irpex lacteus (IlAXE1). IlAXE1 was heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified and characterized. IlAXE1 hydrolyzed p-nitrophenyl acetate, α-naphthyl acetate and 4-methylumbelliferyl acetate, however, it did not show any activity on ethyl ferulate and methyl p-coumarate. We also examined the activity on partially acetylated and feruloylated xylan extracted from corncob by hydrothermal reaction. Similarly, ferulic and p-coumaric acids were not liberated, and acetic acid was only detected in the reaction mixture. The results indicated that IlAXE1 is an acetylxylan esterase actually reacted to acetyl xylan. However, since IlAXE1 was unable to completely release acetic acid esterifying xylopyranosyl residues, it is assumed that acetyl groups exhibiting resistance to deacetylation by IlAXE1 are present in corn cob xylan.     

The viscoelastic properties of soybean curd (tofu) as affected by soymilk concentration and type of coagulant

The viscoelastic properties of different types of tofu were investigated. Soymilk concentrations were 5, 6, 7, 8 and 9%. Coagulants used were 30 mm CaSO₄ or 30 mm glucono‐delta‐lactone (GDL). As the concentration of soymilk was increased viscosity and handling difficulties increased. A high concentration of soymilk in tofu gave a high break stress and produced hard tofu. The four‐element Burgers model fitted the creep behaviour and both viscous and elastic parameters could be acquired from model analysis, reflecting changes in elasticity and viscosity of tofu. The constant viscous parameter in the model increased with increasing soymilk concentration. The viscous parameters of viscoelastic materials like tofu gel, obtained from small deformation tests, seemed to correlate, to some extent, with the break stress obtained from large deformation tests. For hard tofu production increasing the soymilk concentration within a certain range and the partial replacement of calcium sulphate coagulant by GDL could be effective options.     

A comparative study of ethanol production by Issatchenkia orientalis strains under stress conditions

The ability of 13 strains of multi-stress-tolerant Issatchenkia orientalis yeast to produce ethanol was examined under different stress conditions, including conditions of elevated H₂SO₄ and Na₂SO₄ concentrations and increased heat. The MF-121 strain produced a significant amount of ethanol after the incubation in acidic media containing high concentrations of salt, e.g., 50 g/l Na₂SO₄ at pH 2.0, or at high temperatures, e.g., 43°C, when compared with other strains.     

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.     

Development of a strain for efficient degradation of polychlorinated biphenyls by patchwork assembly of degradation pathways

Rhodococcus jostii RHA1 accumulates chlorobenzoates (CBA) during the degradation of polychlorinated biphenyls (PCBs). CBA degradation is considered one of the rate-limiting steps in the complete degradation of PCBs. To reduce the accumulation of CBAs, the upper pathway enzyme genes for PCB degradation of RHA1 were introduced into a CBA-degrading bacterium, Burkholderia sp. NK8. The resulting recombinant strain exhibited no biphenyl 2,3-dioxygenase (BphA) activity encoded by bphAaAbAcAd genes, which encode the large and small subunits of the terminal oxygenase component and the ferredoxin and reductase subunits responsible for electron transfer from NADH to the large subunit. The remaining enzyme genes involved in the transformation of biphenyl to benzoate, bphB2C1D1, which encode dehydrogenase, ring-cleavage dioxygenase and hydrolase, conferred activities to NK8. To obtain the BphA activity of RHA1 in NK8, sets of BphA genes were constructed by combining the bphAaAbAcAd genes of RHA1 and bphA3A4 of Pseudomonas pseudoalcaligenes KF707, encoding the ferredoxin and reductase subunits. Hybrid derivatives of BphA containing the KF707 bphA3 conferred BphA activity to NK8, and a derivative containing the RHA1 bphAaAb and KF707 bphA3A4 genes exhibited the highest BphA activity. A plasmid containing the RHA1 bphAaAb and KF707 bphA3A4 genes plus the RHA1 bphB2C1D1 genes was constructed and introduced into NK8. The resulting recombinant strain efficiently degraded 2-, 3- and 4-chlorobiphenyls with an apparent reduction in CBA accumulation in comparison to the recombinant mutant strain, which had an insertion in the cbeA gene to inactivate CBA dioxygenase.     

The Emi2 Protein of Saccharomyces cerevisiae is a Hexokinase Expressed under Glucose Limitation

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg ²⁺ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.     

Cloning and heterologous expression of a beta-fructofuranosidase gene from Arthrobacter globiformis IFO 3062, and site-directed mutagenesis of the essential aspartic acid and glutamic acid of the active site

We have cloned the gene encoding a beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and subsequently, the gene was heterologously expressed in Escherichia coli. This beta-fructofuranosidase gene encodes a protein of 548 amino acid residues with a calculated molecular mass of 60,519 Da. We have examined the roles of three residues of A. globiformis IFO 3062 beta-fructofuranosidase by site-directed mutagenesis, and found that aspartic acid 130 and glutamic acid 392, which are two of the apparent catalytic residues, are essential for hydrolase activity. This study provides the first experimental evidence showing that these two amino acid residues of beta-fructofuranosidase play a critical role in hydrolyzing sucrose.     

Transmission capacity of longitudinal power systems and SVC voltage control

Power transfer between systems is important due to uneven distribution of generating plants. This paper investigates the relation between the transfer capacity of a longitudinal power system and voltage control of static var compensators. The transfer capacity is basically limited by the thermal capacity of transmission lines. However, the practical systems, it is much restricted by stability and the power transfer level is considerably lower than the thermal capacity. In this paper, we consider a basic case in which SVCs are applied to all buses except generator terminals. In this case, it is possible to transfer power up to the reciprocal of the transmission reactance. Two modifications are then applied to the basic case. One is removal of SVCs on the high-voltage sides of the generator transformers. In this case, generator damping torques deteriorate, and the local oscillation mode becomes unstable. The other is removal of SVCs at intermediate buses on the trunk system. In this case, the shapes of the oscillation modes change greatly, and the global mode becomes unstable. The voltage control of SVCs maintains the generator damping torques and prevents deformation of mode shapes. By investigating different system sizes and transmission circuits, we show that the system transfer capacity is determined by the capacities of the individual transmission lines. © 1997 Scripta Technica, Inc. Electr Eng Jpn, 119(3): 49–60, 1997     

Fault-surge calculation using phase-domain ARMA line model

For fault-surge studies, the most important and also most difficult part of the simulation is inclusion of the frequency dependence of the transmission line, because the simulation requires high accuracy in a wide frequency range, from the power frequency to a few megahertz. The authors have developed a highly efficient method of modeling a transmission line considering its complete frequency dependence, which cannot be dealt with in the present line models in the Electro-Magnetic Transients Program (EMTP). According to the method, a transmission line is modeled directly in the phase domain in order to avoid model transformation and thus to eliminate the problems of representing its frequency dependence. The method also includes sophisticated acceleration of transient calculations by means of an autoregressive moving average (ARMA) model. This paper presents a method of steady-state initialization of the phase-domain ARMA line model for applying it to fault-surge calculations. The calculated results agree well with rigorous frequency-domain simulations and show improved accuracy compared with an existing frequency-dependent line model (J. Marti model) widely used as a standard model in the EMTP. © 1998 Scripta Technica, Inc. Electr Eng Jpn, 121(3): 27–35, 1997