Expression and Characterization of Recombinant Sucrose Phosphorylase

SPase is widely used in the food, cosmetics, and pharmaceutical industries. Previously, a SPase gene was cloned from Bifidobacterium longum JCM1217 and constructed into Escherichia coli BL21. In this paper, its expression conditions were optimized. The results showed that several induction factors determined the expression efficiency of SPase. The initial cell density, IPTG concentration, and induction time and temperature significantly (p?<?0.01) affected the total protein content and activity of expressed SPase. The highest expression efficiency was obtained at an initial cell density of OD₆₀₀?=?0.5, with 0.05 mM IPTG, followed by shaking at 180 rpm and incubation at 30 °C for 15 h. The purified SPase had a specific activity of 122.1 U/mg, which was raised by 1.85 -fold more than that before optimization, and its recovery yield was 86%. Furthermore, SPase also showed higher thermostability. The results of this study provide essential information for the industrial production of SPase.     

Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk

A one-pot enzymatic reaction to produce lacto-N-biose I (LNB), which is supposed to represent the bifidus factor in human milk oligosaccharides, was demonstrated. Approximately 500 mM of LNB was generated in 10-liter of reaction mixture initially containing 660 mM of sucrose and 600 mM of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose-hexose-1-phospate uridylyltransferase, UDP-glucose 4-epimerase, and lacto-N-biose phosphorylase, in the presence of UDP-Glc and phosphate, indicating a reaction yield of 83%. LNB was isolated from the mixture by crystallization after yeast treatment. Finally, 1.4 kg of LNB of 99.6% purity was recovered after recrystallization.     

Efficient isolation of human metapneumovirus using MNT‐1, a human malignant melanoma cell line with early and distinct cytopathic effects

Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC‐MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT‐1, obvious syncytium formation occurs shortly after inoculation with HMPV‐positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT‐1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation‐based surveillance. MNT‐1 has the potential to facilitate clinical and epidemiological studies of HMPV.

     

Activin-A: a modulator of multiple types of anterior pituitary cells

When primary culture of rat pituitary cells were incubated with 1 nM activin-A for more than 24 hrs, activin-A significantly increased intracellular content of FSH without affecting the control of LH. Pretreatment of the cells with activin-A also enhanced LHRH-induced FSH release without affecting LH release. Furthermore, pretreatment of the cells with activin-A significantly reduced both GRF-mediated GH release and TRH-mediated PRL release. However, activin-A did not affect the response of ACTH and TSH to their releasing hormones. These results indicate that, in addition to the known action on gonadotrophs, activin-A also modifies the function of somatotrophs and lactotrophs.     

Stimulation of FSH release by erythroid differentiation factor (EDF)

The action of erythroid differentiation factor (EDF) on primary culture of rat anterior pituitary cells was examined. EDF stimulates FSH secretion in a dose dependent manner but not of LH secretion. ED50 of EDF for FSH secretion was 5 X 10(-10) M, while ED50 of LHRH for FSH secretion was 5 X 10(-9) M. These data indicate that EDF is a potent agonist for FSH secretion and the biological activity of EDF on anterior pituitary seems to be identical as that of FSH releasing protein (FRP).     

A family 8 glycoside hydrolase from Bacillus halodurans C-125 (BH2105) is a reducing end xylose-releasing exo-oligoxylanase

The gene encoding family 8 glycoside hydrolases from Bacillus halodurans C-125 (BH2105), an alkalophilic bacterium with a known genomic sequence, was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it did not possess a signal peptide and that it was an intracellular enzyme. The recombinant enzyme showed no hydrolytic activity on xylan, whereas it had been annotated as xylanase Y. It hydrolyzed xylooligosaccharide whose degree of polymerization is greater than or equal to 3 in an exo-splitting manner with anomeric inversion, releasing the xylose unit at the reducing end. Judging from its substrate specificity and reaction mechanism, we named the enzyme reducing end xylose-releasing exo-oligoxylanase (Rex). Rex was found to utilize only the beta-anomer of the substrate to form beta-xylose and alpha-xylooligosaccharide. The optimum pH of the enzymatic reaction (6.2-7.3) was found in the neutral range, a range beneficial for intracellular enzymes. The genomic sequence suggests that B. halodurans secretes two endoxylanases and possesses two alpha-arabinofuranosidases, one alpha-glucuronidase, and three beta-xylosidases intracellularly in addition to Rex. The extracellular enzymes supposedly hydrolyze xylan into arabino/glucurono-xylooligosaccharides that are then transported into the cells. Rex may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides.     

NADH-Dependent Glutamat Synthase in Euglena gracilis z

Novel antioxidative phenylpropanoid-substituted tocopherol derivatives, prunusols A and B, were isolated from the leaf wax of Prunus grayana Maxim., and their structures were fully characterized by spectroscopic and synthetic methods. Prunusols A and B were found to be the conjugates of γ-tocopherol and p-coumaric acid, which are diastereoisomers of each other. They showed almost the same antioxidative activity as α-tocopherol in a water/alcohol system measured by thiocyanate and TBA methods.     

Subcellular Location and Some Properties of Isocitrate Dehydrogenase Isozymes in Euglena gracilis

Under thiamine-deficient, aerobic culture conditions, Yarrowia lipolytica was found to produce D-(+)-2-hydroxyglutaric acid extracellularly in amounts of about 5 mg per ml of the culture filtrate, together with pyruvic and 2-ketoglutaric acids, from glucose or glycerol in a chemically defined medium. Under similar conditions, however, only a small amount of the hydroxy acid was produced from odd- or even-numbered n-alkanes.     

Purification and Immunological Properties of NAD+-linked Malate Dehydrogenase Isozymes from Euglena gracilis

We investigated the hypolipidemic effects of young persimmon fruit (YP) on apolipoprotein E-deficient C57BL/6.KOR-ApoE^shl mice. These mice exhibited higher plasma cholesterols, except for high-density lipoprotein (HDL), and lower plasma HDL cholesterol than C57BL/6.Cr mice that had the same genetic background as the C57BL/6.KOR-ApoE^shl mice. Male C57BL/6.KOR-ApoE^shl mice (n=5) were fed a diet supplemented with dry YP, Hachiya-kaki, at a concentration of 5% (w/w) for 10 weeks. YP treatment significantly lowered plasma chylomicron, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) cholesterols, and triglyceride, and this response was accompanied by an elevation of fecal bile acid excretion. In the liver, sterol regulatory element binding protein-2 gene expression was significantly higher in mice fed YP, while the mRNA and protein levels of the LDL receptor did not change. These results indicate that acceleration of fecal bile acid excretion is a major mechanism of the hypolipidemic effect induced by YP in C57BL/6.KOR-ApoE^shl mice.     

Growth Inhibition by Phenylalanine in Euglena gracilis

The hydrolysis and esterification by a thermostable lipase from Humicola lanuginosa No. 3 were investigated. Both reactions occurred readily at temperatures between 45~50°C. Esterification by the enzyme with glycerol was observed to be specific towards fatty acids with carbon numbers of C12~C18. Laurie acid esters with different alcohols such as primary alcohols, terpene alcohols, eie., were also synthesized readily. Esterification by the enzyme was adversely affected by the water content (optimum, ca. 7%), however, the hydrolysis rate increased rapidly with increasing water content (optimum, az. 60%). The enzyme showed increased activity in organic solvent-aqueous reaction systems. Nevertheless, hydrolysis in complete organic phase reactions was found not to be feasible. Hydrolysis at a higher temperature (50 or 55°C) in a solvent free phase was almost the same as that in organic solvent-aqueous phase reactions. The components of glycerides varied considerably during hydrolysis, whereby esterification resulted in a higher quantity of mono- and diglycerides (about 40%), compared to in the case of hydrolysis, for which the value was about 10~20%.