Enoch Y. Park

Waste bleaching earths from crude vegetable oil refining process contain approximately 40% of its weight as oil. Low valued oils are potential substrates for biodiesel fuel production. Vegetable oils from waste bleaching earth samples were organic-solvent extracted and identified as ...

The enhancement of -poly--lysine ( -PL) production by Streptomyces albulus strain no. 410 (S410) by means of a pH control strategy was investigated. S140 cells produce -PL at a high concentration if the culture pH remains at about 4.0; however, if it shifts to higher than 4.0, ...

Production of fatty acid alkyl esters by lipase-catalyzed alcoholysis of waste plant oil from oil processing industry with primary alcohols in an organic solvent system was investigated. The catalytic activity of several commercial lipases on waste vegetable oil absorbed in ...

The effects of protease inhibitors on the production of recombinant protein were investigated using a recombinant baculovirus containing GFPuv-human beta 1,3-N-acetylglucosaminyltransferase 2 (beta 3GnT2) connected to the prepromelittin signal sequence. The addition of leupeptin as a cysteine protease inhibitor at 2.5 microg/ml improved intra- and extracellular beta 3GnT activities 5- and 3-fold, respectively, compared to those without addition, which was due to a suppression of protease activity. With the leupeptin addition only four degraded molecular bands lower than 32 kDa appeared, but 9 degraded molecular bands between 29 kDa and 41 kDa existed without addition. In contrast, pepstatin A as a carboxyl protease inhibitor had no influence on the improvement of beta 3GnT production, judging from SDS-PAGE. Moreover, when 50 microM carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), known as a proteasome inhibitor, was used in combination with the leupeptin, a ladder of low molecular mass bands of fusion protein was diminished. The intracellular beta 3GnT activity increased 9-fold, to as high as that without addition of two kinds of protease, but the extracellular activity was not different from that with the addition of only leupeptin. These findings indicate that the decrease in cell viability causes the decrease in the secretion rate of intracellular fusion protein, resulting the accumulation of the full-length of fusion protein.

Active beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. beta3GnT2 was expressed as a secreted fusion protein with GFP(UV) with three different types of signal sequence to enhance the secretion of the fusion protein. In the stably transformed cells, the maximal beta3GnT2 activity differed between isolates, but their secretion efficiencies were similar. The difference between the maximal beta3GnT activities of the isolates studied was considered to be due to the presence of a copy number of the fusion gene, as determined on the basis of the results of Southern blot analysis. The beta3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a beta3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% on SDS-PAGE, in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, beta3GnT2.

Fatty acid methyl ester (FAME) production from waste activated bleaching earth (ABE) discarded by the crude oil refining industry was investigated using fossil fuel as a solvent in the esterification of triglycerides. Lipase from Candida cylindracea showed the highest stability in diesel oil. Using diesel oil as a solvent, 3 h was sufficient to obtain a yield of approximately 100% of FAME in the presence of 10% lipase from waste ABE. Kerosene was also a good solvent in the esterification of triglycerides embedded in the waste ABE. Fuel analysis showed that the FAME produced using diesel oil as a solvent complied with the Japanese diesel standard and the 10% residual carbon amount was lower than that of FAME produced using other solvents. Use of diesel oil as solvent in the FAME production from the waste ABE simplified the process, because there was no need to separate the organic solvent from the FAME-solvent mixture. These results demonstrate a promising reutilization method for the production of FAME, for use as a biodiesel, from industrial waste resources containing waste vegetable oils.

Long-term behavior of rapeseed oil in waste activated bleaching earth (ABE) and the effect of this oil on riboflavin production in the culture of Ashbya gossypii were investigated. Waste ABE with 40% (w/w) rapeseed oil was stored for 80 d, and the extent of oxidation of rapeseed oil was measured by several analytical methods to determine the chemical properties of the oil at different stages of the oil deterioration process:peroxide value, acid value, concentrations of organic acids, acetaldehyde and unsaturated fatty acid, and content of polymerized triglycerides. Peroxide value, acid value, and concentrations of organic acids and acetaldehyde did not affect riboflavin production. However, the content of polymerized triglycerides markedly increased the viscosity of rapeseed oil and was the main reason for the exponential decrease in riboflavin production. A good correlation between the polymerized triglyceride content or viscosity and riboflavin production in the culture of A. gossypii using rapeseed oil as the sole carbon source was found.

1M 7YR /[SR 8EOEWLM (SNMQE ERH )RSGL = 4EVO (epartment of %pplied Biological 'hemistry, *aculty of %griculture, Shizuoka 9niversity, 836 Ohya, Shizuoka 422 8529, Japan Production 8echnology Laboratory, Kaken Pharmaceutical 'o. Ltd., 301 +ensuke, *ujieda 426 8646, ...

A series of spacer-N-linked glycopolymers carrying long/short α2,3/6 sialylated glycan were designed as polymeric inhibitors of influenza virus. Lactose (Lac) and N-acetyllactosamine (LN: Galβ1,4GlcNAc) were first converted to spacer-N-linked disaccharide glycosides, followed by consecutive enzymatic addition of GlcNAc and Gal residues to the glycosides. The resulting spacer-N-linked glycosides with di-, tetra-, and hexasaccharides carrying a Lac, LN, lacto-N-neotetraose (LNnT: Galβ1,4GlcNAcβ1,3Galβ1,4Glc), and LNβ1,3LNnT were coupled to the carboxy group of γ-polyglutamic acid (γ-PGA) and enzymatically converted to glycopolypeptides carrying α2,3/6 sialylated glycans. The interactions of a series of sialoglycopolypeptides with avian and human influenza virus strains were investigated using a hemagglutination inhibition assay. The avian virus A/Duck/HongKong/313/4/78 (H5N3) bound specifically, regardless of the structure of the asialo portion. In contrast, human virus A/Aichi/2/68 (H3N2) bound preferentially to long α2,6sialylated glycans with penta- or heptasaccharides in a glycan length-dependent manner. Furthermore, the Sambucus sieboldiana (SNA) lectin was also useful as a model of human virus hemagglutinin (HA) for understanding the carbohydrate binding properties, because the recognition motifs of the inner sugar in the receptor were very similar.

We designed a series of gamma-polyglutamic acid (gamma-PGA)-based glycopolypeptides carrying long/short alpha2,3/6 sialylated glycans to act inhibitors of the influenza virus. As an alternative design, sialoglycopolypeptides carrying long-spacer linked glycans were engineered by replacement of the N-acetyllactosamine (LN) unit by an alkyl chain. The structure-activity relationship of the resulting sialoglycopolypeptides with different glycans in the array has been investigated by in vitro and in vivo infection experiments. The avian viruses specifically bound to glycopolypeptides carrying a short sialoglycan with higher affinity than to a long glycan. In contrast, human viruses, preferentially bound not only to long alpha2,3/6 sialylated glycan with LN repeats in the receptors, but also to more spacer-linked glycan in which the inner sugar has been replaced by a nonsugar structural unit such as a pentylamido group. Taken together, our results indicate that a spaced tandem/triplet pentylamido repeat is a good mimetic of a tandem/triplet LN repeat. Our strategy provides a facile way to design strong polymeric inhibitors of infection by avian and human influenza viruses.