Purification, characterization and comparison of phycoerythrins from three different marine cyanobacterial cultures

The present study is focused on purification, characterization and comparison of phycoerythrins from three different marine cyanobacterial cultures--hormidium sp. A27 DM, Lyngbya sp. A09 DM and Halomicronema sp. A32 DM. 'Phycoerythrin' was successfully purified and characterized. On SDS-PAGE, the PE purified from all three young cultures showed four bands--corresponding to α and β subunits of each of PE-I and PE-II. However, phycoerythrin purified after prolonged growth of Phormidium sp. A27 DM and Halomicronema sp. A32DM showed only one band corresponding to 14 kDa whereas Lyngbya sp. A09 DM continued to produce uncleaved phycoerythrin. The absorption spectra of purified PEs from all the three young and old cultures showed variations however the fluorescence studies of the purified PEs in all cases gave the emission spectra at around 580 nm. The described work is of great importance to understand the role of phycoerythrin in adapting cyanobacteria to stress conditions.     

Chromatographic separation and spectroscopic characterization of the e/z isomers of acrivastine

A reverse phase high performance liquid chromatography (HPLC) method has been developed for the separation of two geometric isomers of Acrivastine using crude reaction mixture. The resolution between two isomers was found more than 2.9. The geometric isomers have been isolated by preparative HPLC and characterized by spectroscopic techniques, such as NMR, infrared, and MS. The developed method has been validated for the determination of Z‐isomer in Acrivastine. The limit of detection and limit of quantification of the Z‐isomer were 0.05 and 0.2 μg/ml, respectively. The developed method is precise, linear, accurate, rugged and robust for its intended use. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Biotechnological interventions in sea buckthorn (Hippophae L.): current status and future prospects

Sea buckthorn (Hippophae L., Elaeagnaceae) is an economically and ecologically important medicinal plant comprising of species which are winter hardy, dioecious, wind-pollinated multipurpose shrubs bearing yellow or orange berries with nitrogen-fixing ability. It grows widely in cold regions of Indian Himalayas, China, Russia, Europe and many other countries. It is commonly known as ‘cold desert gold’ due to its high potential as a bio-resource for land reclamation, reducing soil erosion and its multifarious uses. The wild populations are being used for harvesting economic benefits with negligible plantation efforts. Although this plant has many excellent traits, it is still in an early phase of domestication. This woody plant is prone to many pests and diseases which destroy the plants and halt its commercial production. Limited progress has been made for improvement of sea buckthorn through breeding programs due to long juvenile period and lack of QTL linkage map, which makes screening of mapping populations a time-consuming and labor-intensive task. Conventional propagation methods, i.e. seeds, softwood and hardwood cuttings, and suckers are in place but are cumbersome and season dependent. Therefore, application of modern tools of biotechnology needs to be standardized for harnessing maximum benefits from this nutraceutical plant. Improvement of this genus through genetic transformation requires an efficient regeneration system, which is yet to be standardized. Taxonomic status of the genus is controversial and requires more inputs. Taxonomic delineation of species and subspecies and also the breeding programs can be more robustly addressed using molecular markers. This review summarizes the progress made and suggests some future directions of research for this important fruit species.     

Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.     

Purification and partial characterization of an extracellular alginate lyase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> isolated from brown seaweed

The extracellular enzyme alginate lyase produced from marine fungus Aspergillus oryzae isolated from brown alga Dictyota dichotoma was purified, partially characterized, and evaluated for its sodium alginate depolymerization abilities. The enzyme characterization studies have revealed that alginate lyase consisted of two polypeptides with about 45 and 50 kDa each on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed 140-fold higher activity than crude enzyme under optimized pH (6.5) and temperature (35°C) conditions. Zn²⁺, Mn²⁺, Cu²⁺, Mg²⁺, Co²⁺ and NaCl were found to enhance the enzyme activity while (Ca²⁺, Cd²⁺, Fe²⁺, Hg²⁺, Sr²⁺, Ni²⁺), glutathione, and metal chelators (ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid) suppressed the activity. Fourier transform infrared and thin-layer chromatography analysis of depolymerized sodium alginate indicated the enzyme specificity for cleaving at the β-1,4 glycosidic bond between polyM and polyG blocks of sodium alginate and therefore resulted in estimation of relatively higher polyM content than polyG. Comparison of chemical shifts in ¹³C nuclear magnetic resonance spectra of both polyM and polyG from that of sodium alginate also showed further evidence for enzymatic depolymerization of sodium alginate.     

Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate

The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k(cat)/K(M) up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity.     

Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by calcified elastic fibers in cutaneous, ocular and vascular tissues. PXE is caused by mutations in ABCC6, which encodes a protein of the ATP-driven organic anion transporter family. The inability of this transporter to secrete its substrate into the circulation is the likely cause of PXE. Vitamin K plays a role in the regulation of mineralization processes as a co-factor in the carboxylation of calcification inhibitors such as Matrix Gla Protein (MGP). Vitamin K precursor or a conjugated form has been proposed as potential substrate(s) for ABCC6. We investigated whether an enriched diet of vitamin K1 or vitamin K2 (MK4) could stop or slow the disease progression in Abcc6^-/- mice. Abcc6^-/- mice were placed on a diet of either vitamin K1 or MK4 at 5 or 100 mg/kg at prenatal, 3 weeks or 3 months of age. Disease progression was quantified by measuring the calcium content of one side of the mouse muzzle skin and histological staining for calcium of the opposing side. Raising the vitamin K1 or MK4 content of the diet increased the concentration of circulating MK4 in the serum. However, this increase did not significantly affect the MGP carboxylation status or reduce its abnormal abundance, the total calcium content or the pathologic calcification in the whiskers of the 3 treatment groups compared to controls. Our findings showed that raising the dietary intake of vitamin K1 or MK4 was not beneficial in the treatment of PXE and suggested that the availability of vitamin K may not be a limiting factor in this pathology.Key words: pseudoxanthoma elasticum, vitamin K, mineralization, Abcc6, mouse