Surfactant-Modified lipase for the catalysis of the interesterification of triglycerides and fatty acids

The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. (c) 1995 John Wiley & Sons, Inc.     

Distributions of larval mosquitoes among bamboo-stump pools which vary in persistence and resource input

We studied the seasonal occurrence and the distribution patterns of larval mosquitoes in a bamboo grove in northern Kyushu, Japan. The number of pools was large from June to August and was small in winter. Deep stumps and semi-closed-type stumps held water more persistently than shallow and open-type stumps, respectively. Open-type stumps trapped more leaves than semi-closed-type stumps and the number of leaves trapped in the open-type stump was positively correlated with the area of the pool. The incidences ofTripteroides bambusa andAedes albopictus were low early in their breeding season and gradually increased thereafter. In August, the density ofT. bambusa larvae per pool was higher in the old (>2 months) pools than in the new pools and was positively correlated with the depth of the stumps. In contrast, the density ofA. albopictus did not differ significantly between the new-and old pools and was not significantly correlated with the depth of the stump. The density ofA. albopictus was positively correlated with the number of leaves that had been trapped in the stump, whereas the density ofT. bambusa was not. In addition to the seasonal fluctuation in habitat-patch availability, the variations in habitat persistence and resource input among bamboo stumps may facilitate the coexistence of the two mosquitoes that were differentially limited by these factors.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

Intracellular localization of the arginine deiminase pathway inStreptococcus mitis

A subcellular fractionation study was carried out onStreptococcus mitis ATCC 9811 to determine the location of the arginine deiminase pathway. Arginine deiminase activity was detected in the cell wall fraction, ornithine carbamoyltransferase activity was recovered in both the cell wall and cytoplasmic fractions, and carbamate kinase activity was detected in the cytoplasmic fraction.     

Effects of amino acid substitutions in the F helix of bacteriorhodopsin. Low temperature ultraviolet/visible difference spectroscopy

Site-specific mutagenesis in combination with low temperature UV/visible difference spectroscopy has been used to investigate the role of individual amino acids in the structure and function of bacteriorhodopsin (bR). We examined the effects of eight single amino acid substitutions, all in the putative F helix, on the absorption of bR as well as formation of the K and M intermediates. Both the absorbance spectra and the photocycle difference spectra of Escherichia coli expressed bR as well as the mutants S183A, P186G, and E194Q all closely resembled the corresponding purple membrane spectra. In contrast the Pro-186----Leu substitution resulted in the loss of the normal photocycle and a large blue shift in the bR state lambda max. Thus, Pro-186 appears to play a critical role in maintaining the normal protein-chromophore interactions, although the pyrrolidine ring is not essential since proline could be replaced by glycine at this position. The mutants W182F, W189F, and S193A did not appear to be directly involved in the bathochromic shift of bR since they all had lambda max's close to that of purple membrane and produced intermediates similar to K and M. However, alterations in the UV and visible difference spectra as well as the appearance of some irreversibility in the photoreactions indicate that these mutants have altered protein-chromophore interactions during the photocycle. Unlike the other mutants examined, Y185F exhibited a red-shifted form of bR and K raising the possibility that Tyr-185 is directly involved in color regulation. In addition, UV difference peaks previously associated with a tyrosine deprotonation were absent in Y185F indicating that Tyr-185 undergoes protonation changes during the photocycle in agreement with recent Fourier transform infrared difference measurements (Braiman, M.S., Mogi, T., Stern, L. J., Hackett, N., Chao, B. H., Khorana, H.G., and Rothschild, K. J. (1988) Proteins: Structure, Function, and Genetics 3, 219-229). Our results suggest that Trp-182, Tyr-185, Pro-186, Trp-189, and Ser-193, all of which are within a 100 degrees segment of the F helix, are part of a retinal-binding pocket.     

Vibrational spectroscopy of bacteriorhodopsin mutants: chromophore isomerization perturbs tryptophan-86

Fourier transform infrared difference spectra have been obtained for the bR----K and bR----M photoreactions of bacteriorhodopsin mutants with Phe replacements for Trp residues 10, 12, 80, 86, 138, 182, and 189 and Cys replacements for Trp residues 137 and 138. None of the tryptophan mutations caused a significant shift in the retinylidene C = C or C-C stretching frequencies of the visible absorption maximum of the chromophore, it is concluded that none of the tryptophan residues are essential for forming a normal bR570 chromophore. However, a 742-cm-1 negative peak attributed previously to the perturbation of a tryptophan residue during the bR----K photoreaction was found to be absent in the bR----K and bR----M difference spectra of the Trp-86 mutant. On this basis, we conclude that the structure or environment of Trp-86 is altered during the bR----K photoreaction. All of the other Trp----Phe mutants exhibited this band, although its frequency was altered in the Trp-189----Phe mutant. In addition, the Trp-182----Phe mutant exhibited much reduced formation of normal photoproducts relative to the other mutants, as well as peaks indicative of the presence of additional chromophore conformations. A model of bR is discussed in which Trp-86, Trp-182, and Trp-189 form part of a retinal binding pocket. One likely function of these tryptophan groups is to provide the structural constraints needed to prevent chromophore photoisomerization other than at the C13 = C14 double bond.     

Identification of new inhibitors for alternative NADH dehydrogenase (NDH-II)

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans , we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.