Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor

Ethyl acetate was explored as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel from the crude oils of Jatropha curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The optimum reaction conditions for interesterification of the oils with ethyl acetate were 10% of Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, ethyl acetate to oil molar ratio of 11:1 and the reaction period of 12h at 50 degrees C. The maximum yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils, respectively under the above optimum conditions. Reusability of the lipase over repeated cycles in interesterification and ethanolysis was also investigated under standard reaction conditions. The relative activity of lipase could be well maintained over twelve repeated cycles with ethyl acetate while it reached to zero by 6th cycle when ethanol was used as an acyl acceptor.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

The sites for catalysis and activation of ribulosebisphosphate carboxylase share a common domain

The complexation of ribulosebiphosphate carboxylase with CO2, Mg2+, and carboxyarabinitol bisphosphate (CABP) to produce the quaternary enzyme-carbamate-Mg2+-CABP complex closely mimics the formation of the catalytically competent enzyme-carbamate-Mg2+-3-keto-CABP form during enzymatic catalysis. Quaternary complexes were prepared with various metals (Mg2+, Cd2+, Mn2+, Co2+, and Ni2+) and with specifically 13C-enriched ligands. 31P and 13C NMR studies of these complexes demonstrate that the activator CO2 site (carbamate site), the metal binding site, and the substrate binding site are contiguous. It follows that both the carboxylase and oxygenase activities of this bifunctional enzyme are influenced by the structures of the catalytic and activation sites.     

Rapid resolution of phenylthiohydantoin amino acids by thin-layer chromatography on silica gel plates impregnated with transition metal ions

The resolution of a 15-component mixture of phenylthiohydantoin (PTH) amino acids using metal ion impregnated silica gel plates is reported. The spots are located by exposing the chromatograms to an iodine chamber. The method provides a rapid, simple, and less expensive chromatographic system, provides resolution for certain difficult combinations, and leaves the PTH amino acids unaltered chemically.     

Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium

An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene.     

QTL analysis for grain quality traits in 2 BC2F2 populations derived from crosses between Oryza sativa cv Swarna and 2 accessions of O. nivara

The appearance and cooking quality of rice determine its acceptability and price to a large extent. Quantitative trait loci (QTLs) for 12 grain quality traits were mapped in 2 mapping populations derived from Oryza sativa cv Swarna × O. nivara. The BC(2)F(2) population of the cross Swarna × O. nivara IRGC81848 (population 1) was evaluated during 2005 and that from Swarna × O. nivara IRGC81832 (population 2) was evaluated during 2006. Linkage maps were constructed using 100 simple sequence repeat (SSR) markers in population 1 and 75 SSR markers in population 2. In all, 21 QTLs were identified in population 1 (43% from O. nivara) and 37 in population 2 (38% QTLs from O. nivara). The location of O. nivara-derived QTLs mp1.2 for milling percent, kw6.1 for kernel width, and klac12.1 for kernel length after cooking coincided in the 2 populations and appear to be useful for Marker Assisted Selection (MAS). Four QTLs for milling percent, 1 QTL each for amylose content, water uptake, elongation ratio, 2 QTLs for kernel width, and 3 QTLs for gel consistency, each explained more than 20% phenotypic variance. Three QTL clusters for grain quality traits were close to the genes/QTLs for shattering and seed dormancy. QTLs for 4 quality traits were associated with 5 of the 7 major yield QTLs reported in the same 2 mapping populations. Useful introgression lines have been developed for several agronomic traits. It emerges that 40% O. nivara alleles were trait enhancing in both populations, and QTLs for grain quality overlapped with yield meta-QTLs and QTLs for dormancy and seed shattering.     

Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, -, - and -catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.     

Ca-binding and Ca-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca²⁺. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca²⁺ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10⁶ M⁻¹, and 21.58·10⁻⁶ moles·g⁻¹. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca²⁺ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca²⁺. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.