Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Inhibitory effect of amines on polar filament extrusion byPlistophora anguillarum spores

In in vitro tests, amines were screened for the inhibition of polar filament extrusion by spores ofPlistophora anguillarum, a microsporidian parasite of the eel. Primary amines having C₈ ⁻ C₁₈ alkyl chains were effective, irrespective of the anions bound. Secondary amines having a branched C₈ chain and tertiary amines having C₁₄−C₁₈ chains were also effective. Asymmetry in side chains seemed to be required for inhibitory action. Quaternary ammonium salts having C₁₂−C₁₆ alkyl chains and some germicides or disinfectants such as cetyl pyridinium chloride and benzethonium chloride were also effective. Diamines and amides were ineffective regardless of the length of their alkyl chins. In in vivo tests, eel larvae-fed for 4 days with a commercial feed supplemented with 5×10⁸ cells of spores and 4g of laurylamine aspartate/100 g feed, and thereafter with the usual commercial feed for 26 days—were not infected.     

l-Serine Production Improved by Analogue-resistant Mutants of a Methanol-utilizing Bacterium

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

It has been found that although Brevibacterium lactofermentum No. 2256 is incapable of accumulating l-glutamic acid in a biotin sufficient medium, it produces a large quantity of the acid in the presence of sucrose fatty acid ester. In a biotin deficient medium, however, the ester brought the unfavorable diminution of l-glutamic acid accumulation caused by the decrease of glucose consumption in an incubation period. The undesirable effects were practically lost when the ester was added to the culture medium after more than eight hours in the course of incubation. This fact suggests that the ester is concerned with the growth of microorganism. It is very interesting to elucidate the interrelation between sucrose fatty acid ester and biotin. For the maximum accumulation of l-glutamic acid corresponding increase in amount of the ester to the increasing concentration of biotin was necessary. The proportional relation did not extend to excedingly high levels of the two implicating factors. The further observations concerning the effects of the individual fatty acid esters such as sucrose stearate remain unsatisfactory.     

Genetic isolation between the Western and Eastern Pacific populations of pronghorn spiny lobster Panulirus penicillatus

The pronghorn spiny lobster, Panulirus penicillatus, is a circumtropical species which has the widest global distribution among all the species of spiny lobster, ranging throughout the entire Indo-Pacific region. Partial nucleotide sequences of mitochondrial DNA COI (1,142-1,207 bp) and 16S rDNA (535-546 bp) regions were determined for adult and phyllosoma larval samples collected from the Eastern Pacific (EP)(Galápagos Islands and its adjacent water), Central Pacific (CP)(Hawaii and Tuamotu) and the Western Pacific (WP)(Japan, Indonesia, Fiji, New Caledonia and Australia). Phylogenetic analyses revealed two distinct large clades corresponding to the geographic origin of samples (EP and CP+WP). No haplotype was shared between the two regional samples, and average nucleotide sequence divergence (Kimura's two parameter distance) between EP and CP+WP samples was 3.8±0.5% for COI and 1.0±0.4% for 16S rDNA, both of which were much larger than those within samples. The present results indicate that the Pacific population of the pronghorn spiny lobster is subdivided into two distinct populations (Eastern Pacific and Central to Western Pacific), with no gene flow between them. Although the pronghorn spiny lobster have long-lived teleplanic larvae, the vast expanse of Pacific Ocean with no islands and no shallow substrate which is known as the East Pacific Barrier appears to have isolated these two populations for a long time (c.a. 1MY).     

A late-stage phyllosoma larva of the spiny lobster Panulirus echinatus Smith, 1869 (Crustacea: Palinuridae) identified by DNA analysis

A late-stage phyllosoma larva of the genus Panulirus was caughtin the central Atlantic in October 2000. Nucleotide sequenceanalysis of mitochondrial 16S rDNA identified this larva asPanulirus echinatus, which has been undescribed to date. Morphologicalexamination indicated that this eighth stage larva belongedto phyllosoma species group 2, distinguishable from the otherAtlantic species of the genus but closely related to an Indo-Pacificspecies, P. penicillatus, which is in accordance with the resultsof a molecular phylogenetic analysis.     

Production of adenine arabinoside by gel-entrapped cells of Enterobacter aerogenes in water-organic cosolvent system

Summary Gel-entrapped whole cells of Enterobacter aerogenes, which has a transglycosylation activity, were used to produce adenine arabinoside from uracil arabinoside and adenine, in an appropriate water-organic cosolvent system. Cells of E. aerogenes entrapped with a hydrophilic photo-crosslinkable resin prepolymer, ENT-4000, or a urethane prepolymer, PU-6, had a high and stable transglycosylation activity. To improve the poor solubility in water of the substrate (adenine) and product (adenine arabinoside), dimethyl sulfoxide was selected as the cosolvent based on the criteria of operational stability of the immobilized biocatalyst and solubility of both substrate and product. Addition of 40% dimethyl sulfoxide to the reaction mixture permitted use of a high substrate concentration range which gave high productivity under homogeneous reaction conditions. The immobilized cells of E. aerogenes exhibited a markedly improved operational stability, retaining their initial level of activity during repeated use for at least 35 days at 60°C in 40% dimethyl sulfoxide. When the reaction was carried out with 150 mM uracil arabinoside and 50 mM adenine as the substrates, the yield of adenine arabinoside was maintained at 100% based on the molar ratio of adenine, throughout the reaction.Abbreviations used AraA adenine arabinoside - AraU uracil arabinoside     

Development of quantitative plasma N-glycoproteomics using label-free 2-D LC-MALDI MS and its applicability for biomarker discovery in hepatocellular carcinoma

There has been rapid progress in the development of clinical proteomic methodologies with improvements in mass spectrometric technologies and bioinformatics, leading to many new methodologies for biomarker discovery from human plasma. However, it is not easy to find new biomarkers because of the wide dynamic range of plasma proteins and the need for their quantification. Here, we report a new methodology for relative quantitative proteomic analysis combining large-scale glycoproteomics with label-free 2-D LC-MALDI MS. In this method, enrichment of glycopeptides using hydrazide resin enables focusing on plasma proteins with lower abundance corresponding to the tissue leakage region. On quantitative analysis, signal intensities by 2-D LC-MALDI MS were normalized using a peptide internal control, and the values linked to LC data were treated with DeView? software. Our proteomic method revealed that the quantitative dynamic ranged from 102 to 10? pg/mL of plasma proteins with good reproducibility, and the limit of detection was of the order of a few ng/mL of proteins in biological samples. To evaluate the applicability of our method for biomarker discovery, we performed a feasibility study using plasma samples from patients with hepatocellular carcinoma, and identified biomarker candidates, including ceruloplasmin, alpha-1 antichymotrypsin, and multimerin-1.     

5′-Nucleotidase Activity in Improved Inosine-producing Mutants of Bacillus subtilis

An inosine- and guanosine-producing strain, AJ11100, of Bacillus subtilis could not grow in the minimum medium supplemented with 50 µg of sulfaguanidine per ml. When sulfaguanidine resistant mutants were derived from AJ11100, the sulfaguanidine resistance was frequently accompanied by xanthine requirement. All the xanthine auxotrophic mutants required a large amount of xanthine for cell growth and inosine accumulation. Revertants were then derived from one of the xanthine auxotrophic mutants, AJ11101, and improved inosine producers were obtained. The best mutant, AJ11102, accumulated 20.6 g of inosine per liter.

Furthermore, enzyme activities of inosine 5′-monophosphate (IMP) dehydrogenase, 5′-nucleotidase and phosphoribosyl pyrophosphate (PRPP) amidotransferase were assayed to investigate why AJ11102 accumulated an increased amount of inosine. The results showed that the increase of specific activity of 5′-nucleotidase contributed much to the increased accumulation of inosine.