Optimization of carotenoid production from hyper-producing Rhodotorula glutinis mutant 32 by a factorial approach

AIMS: Optimization of carotenoid production by a mutant of Rhodotorula glutinis. METHODS: The growth and carotenoid production was optimized in shake flasks using a two-level, three-variable factorial design. RESULTS: The volumetric carotenoid production could be increased to 129 +/- 2 mg x 1(-1) in a medium containing (g x l(-1)) yeast extract 11.74, glucose 46 and threonine 18 along with other micronutrients, wherein, beta-carotene yield was 102 +/- 2 mg x l(-1), accounting for 80% of the total carotenoids. SIGNIFICANCE AND IMPACT OF THE STUDY: The medium optimization resulted in a fourfold increase in volumetric production and a twofold increase in the cellular accumulation of carotenoids. In view of such high yields, the mutant of Rhodotorula glutinis can be a potential source of beta-carotene.     

A new large species of gecko of the genus Hemidactylus Oken, 1817 (Reptilia: Sauria: Gekkonidae) from the Eastern Ghats,India

A new large species of gecko superficially resembling Hemidactylus maculatus Duméril & Bibron and its allied large Hemidactylus is described from Eastern Ghats. The new species, Hemidactylus kangerensis sp. nov. can be easily distinguished from members of the H. maculatus complex based on the number of femoral pores, i.e. 18–21 on each side, separated medially by 4 non-pored scales. A phylogenetic analysis based on partial sequence of mitochondrial gene cytochrome b shows that the gecko is a member of the “H. prashadi” clade and is sister to H. maculatus, from which it differs in an uncorrected pairwise sequence divergence of 10%. The discovery of a new large gecko from Eastern Ghats advocates the need for biodiversity assessment across the neglected Eastern Ghats.     

Phytochemical Profiling,Antimicrobial, Antiproliferative and Apoptotic Effects of Stemodia viscosa Roxb. of Western Ghats Region,India

The present study shows the chemical profile, antimicrobial, antiproliferative, and apoptotic effects of Stemodia viscosa extracts. Thirteen bioactive compounds were identified in the 80 % ethanolic extract by GC/MS analysis. The acetone extract exhibited a higher content of flavonoids and phenols of 805.10 μg QE/mg DW and 89.31 μg GAE/mg DW extracts, respectively. Furthermore, the acetone extract possessed the highest antioxidant activity (IC₅₀=9.96 μg/mL). The 80 % ethanolic extract exhibited significant antimicrobial activity; the highest activity was observed against Staphylococcus aureus with a zone of inhibition of 25±0.51 mm, MIC value of 4 mg/mL, and MBC value of 8 mg/mL. The antiproliferative results revealed the presence of anticancer activity with an IC₅₀=91.562 and 74.362 μg/mL against the B16F10 skin and COLO205 colon cancer cells, respectively. The flow cytometric analysis shows that the plant extracts cause cancer cell death through the induction of apoptosis. Our findings confirmed that Stemodia viscosa is a potential source of biologically active compounds.     

Locomotory behavior, contact inhibition, and pattern formation of 3T3 and polyoma virus-transformed 3T3 cells in culture

The social behavior of 3T3 cells and their polynoma virus-transformed derivative (Py3T3 cells) was examined by time-lapse cinemicrography in order to determine what factors are responsible for the marked differences in the patterns formed by the two cell lines in culture. Contrary to expectations, both cell types have been found to exhibit contact inhibition of cell locomotion. Therefore, the tendency of 3T3 cells to form monolayers and of Py3T3 cells to form crisscrossed multilayers cannot be explained on the basis of the presence versus the absence of contact inhibition. Morevover, with the exception of cell division control, the social behavior of the two cell types is qualitively similar. Both exhibit cell underlapping and, after contact between lamelliopodia, both show inhibition of locomotory activity and adhesion formation. Neither cell type was observed to migrate over the surface of another cell. The two cell types do show quantitative differences in the frequency of underlapping, the frequency with which contact results in inhibition of locomotion, and the proportion of the cell margin that adheres to the substratum. The increased frequency pf Py3T3 underlapping is correlated with the reduced frequency of substratum adhesions, which in turn favors underlapping. On the basis of these observations, it is concluded that the differences in culture patterns are the result of differences in the shapes of the individual cells, such that underlapping, and hence crisscrossing, is favored in Py3T3 cell interactions and discouraged in 3T3 cells.     

Synergistic effects of zeaxanthin and its binding protein in the prevention of lipid membrane oxidation

There is growing evidence that high levels of the macular xanthophyll carotenoids lutein and zeaxanthin may be protective against visual loss due to age-related macular degeneration, but the actual mechanisms of their protective effects are still poorly understood. We have recently purified, identified and characterized a pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye which specifically and saturably binds to the two forms of zeaxanthin endogenously found in the foveal region. In this report, we studied the synergistic antioxidant role of zeaxanthin and GSTP1 in egg yolk phosphatidylcholine (EYPC) liposomes using hydrophilic 2,2'-azobis(2-methyl-propionamidine) dihydrochloride (AAPH) and lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) as lipid peroxyl radical generators. The two zeaxanthin diastereomers displayed synergistic antioxidant effects against both azo lipid peroxyl radical generators when bound to GSTP1. In the presence of GSTP1, nondietary (3R,3'S-meso)-zeaxanthin was observed to be a better antioxidant than dietary (3R,3'R)-zeaxanthin. This effect was found to be independent of the presence of glutathione. Carotenoid degradation profiles indicated that the zeaxanthin diastereomers in association with GSTP1 were more resistant to degradation which may account for the synergistic antioxidant effects.     

Identification and characterization of a Pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye

Uptake, metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs). A membrane-associated XBP was purified from human macula using ion-exchange chromatography followed by gel-exclusion chromatography. Two-dimensional gel electrophoresis showed a prominent spot of 23 kDa and an isoelectric point of 5.7. Using mass spectral sequencing methods and the public NCBI database, it was identified as a Pi isoform of human glutathione S-transferase (GSTP1). Dietary (3R,3'R)-zeaxanthin displayed the highest affinity with an apparent Kd of 0.33 microm, followed by (3R,3'S-meso)-zeaxanthin with an apparent Kd of 0.52 microm. (3R,3'R,6'R)-Lutein did not display any high-affinity binding to GSTP1. Other human recombinant glutathione S-transferase (GST) proteins, GSTA1 and GSTM1, exhibited only low affinity binding of xanthophylls. (3R,3'S-meso)-Zeaxanthin, an optically inactive nondietary xanthophyll carotenoid present in the human macula, exhibited a strong induced CD spectrum in association with human macular XBP that was nearly identical to the CD spectrum induced by GSTP1. Like-wise, dietary (3R,3'R)-zeaxanthin displayed alterations in its CD spectrum in association with GSTP1 and XBP. Other mammalian xanthophyll carrier proteins such as tubulin, high-density lipoprotein, low-density lipoprotein, albumin, and beta-lactoglobulin did not bind zeaxanthins with high affinity, and they failed to induce or alter xanthophyll CD spectra to any significant extent. Immunocytochemistry with an antibody to GSTP1 on human macula sections showed highest labeling in the outer and inner plexiform layers. These results indicate that GSTP1 is a specific XBP in human macula that interacts with (3R,3'S-meso)-zeaxanthin and dietary (3R,3'R)-zeaxanthin in contrast to apparently weaker interactions with (3R,3'R,6'R)-lutein.     

Isoxazole-tethered diarylheptanoid analogs: Discovery of a new drug-like PAR2 antagonist

A new class of isoxazole-tethered diarylheptanoids having characteristic 1,3-syn-diol and 1,3-anti-diol chemophoric moieties, e.g. 4a–d and 5a–c respectively, have been designed and synthesized starting from d-glucose following a stereo-conserved general synthetic strategy. The isoxazole heterocycle was installed using our recently elaborated methodology deploying Magtrieve? as a selective oxidizing agent. Two of these new analogs 4a and 5a exhibited significantly improved in vitro drug-like properties including solubility, metabolic stability, cell permeability and lack of nonspecific cytotoxicity when compared with curcumin-I. In a HEK293 cell-based intracellular calcium [Ca²⁺]_i release assay, 4a and 5a, when tested at 30?μM, inhibited the trypsin agonist induced protease-activated receptor-2 (PAR2) activity by 80% and 70% respectively. IC₅₀ of 4a (SB70) has been determined as 6?μM which is in the same range of current benchmarks for PAR2 antagonists.     

Molecular and industrial aspects of glucose isomerase.

Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the presence of two signature sequences, VXW(GP)GREG(YSTAE)E and (LIVM)EPKPX(EQ)P. The use of an inexpensive inducer in the fermentation medium devoid of Co2+ and redesigning of a tailor-made GI with increased thermostability, higher affinity for glucose, and lower pH optimum will contribute significantly to the development of an economically feasible commercial process for enzymatic isomerization of glucose to fructose. Manipulation of the GI gene by site-directed mutagenesis holds promise that a GI suitable for biotechnological applications will be produced in the foreseeable future.     

Purification and characterization of 5-aminolevulinic acid dehydratase from Methanosarcina barken

Abstract 5-Aminolevulinic acid dehydratase from the archaebacterium Methanosarcina barken resembles the mammalian and yeast enzymes in its activation by Zn²⁺, whereas its activation by K⁺ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni²⁺ which is a component of F₄₃₀, a cofactor present mainly in methanogens. The M _r of 280000 for the native enzyme and 30 000 ± 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of 5-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates.