Cloning, expression, purification and characterization of UMP kinase from Staphylococcus aureus.

Uridine monophosphate kinase (UMPK) an enzyme of de novo biosynthesis catalyses the formation of UDP and it is involved in cell wall and RNA biosynthesis. In the present study UMPK of Staphylococcus aureus ATCC12600 was characterized. Analysis of purified UMPK by gel filtration chromatography on Sephadex G-200 indicated a molecular weight of 150 kDa and exhibited monomeric form with molecular weight of 25 kDa in SDS-PAGE confirming homohexamer nature of UMPK in solution. The enzyme kinetics of UMPK showed K(m) of 2.80 ± 0.1 µM and Vmax 51.38 ± 1.39 µM of NADH/min/mg. The enzyme exhibited cooperative kinetics with ATP as substrate, as GTP decreased this cooperativity and increased affinity for ATP. The UMPK gene was amplified, sequenced (Accession number: FJ415072), cloned in pQE30 vector and overexpressed in Escherichia coli DH5α. The purified recombinant UMPK showed similar properties of native UMPK. The UMPK gene sequence showed complete homology with pyrH gene sequence of all S. aureus strains reported in the database, the 3D structure of S. aureus UMPK built from the deduced amino acid sequence was super imposed with human UMPK (PDB ID: 1TEV) to find out the structural identity using the MATRAS programme gave an RMSD value 4.24 Å indicating very low homology and extensive structural variations with human UMPK structure. Thus, UMPK may be a potential drug target in the development of antimicrobials.

A comparative in vitro study on antifungal and antioxidant activities of Nervilia aragoana and Atlantia monophylla.

Ethyl acetate extract of the whole plant of Nervilia aragoana Gaud. (Orchidaceae) and ethanol extract of the leaves of Atlantia monophylla Linn. (Rutaceae) were evaluated for antifungal and antioxidant activities. At 5 mg/mL concentration of the extracts, the former exhibited more inhibitory activity than the latter against fungi. The order of MIC values for Nervilia aragoana were Saccharomyces cerevisiae (1.4 mg/mL) > Aspergillus niger (1.2 mg/mL) > Aspergillus fumigatus (0.95 mg/mL) > Cryptococcus neoformans (0.75 mg/mL). In the case of Atlantia monophylla values were Cryptococcus neoformans (1 mg/mL) > Candida albicans (0.95 mg/mL) > Aspergillus niger (0.65 mg/mL). TLC-DPPH method assay was carried out to evaluate the antioxidant potential. Further DPPH radical, superoxide, nitric oxide, H(2)O(2) scavenging, and reducing power activities were carried out. N. aragoana (85%) extract exhibited more scavenging activity than that of A. monophylla (66%) by DPPH free radical scavenging method. A. monophylla extract exhibited more superoxide, nitric oxide, H(2)O(2) scavenging activities than that of N. aragoana. The acute toxicity studies of both extracts have shown no mortality rate even up to 3 g/kg body weight in albino rats. Screening for secondary metabolites showed the presence of carbohydrates in both extracts. Flavonoids were found only in the ethyl acetate extract of N. aragoana. Tannins, alkaloids, triterpenoids and steroids were present in A. monophylla. Total phenols present in N. aragoana and A. monophylla were 340 and 560 mg/g extract of gallic acid equivalents, respectively.

Analysis of volatile aroma constituents of wine produced from Indian mango (Mangifera indica L.) by GC-MS.

Volatile aroma compounds are synthesized by wine yeast during wine fermentation. In this study the volatile aroma composition of two varieties of mango wine were determined to differentiate and characterize the wines. The wine was produced from the fruits of two varieties of mango cultivars namely Banginapalli and Alphonso. The volatile compounds formed in mango wine were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Thirty-two volatile compounds in wines were determined of which four were new and unidentified present in lower concentration. Apart from the ethanol (8.5 ± 0.28 and 7.2 ± 0.28% v/v), 1-propanol (54.11 ± 0.33 and 42.32 ± 0.57 mg/l), isobutyl alcohol (102 ± 1.57 and 115.14 ± 2.88 mg/l) and isoamyl alcohol (123 ± 2.88 and 108.40 ± 0.23 mg/1) were found to be the major flavouring higher alcohols in the mango wines produced from the fruits of Banginapalli and Alphonso respectively. Ethyl acetate (35 ± 0.57 and 30.42 ±1.15 mg/l) was the major ester component in both wines produced. Besides, other esters like ethyl octonoate, ethyl hexanoate and ethyl decanoate were also present in the wines. Cyclohexane methanol (1.45 ± 0.11 mg/l) was present only in wine made from Banginapalli and ß-phenylethyl butanoate (0.62 ± 0.01 mg/1) was found only in Alphonso wine. The results demonstrate that the wine prepared from Banginapalli variety had better aroma composition and good taste than that from the Alphonso variety.

High gravity fermentation of sugarcane molasses to produce ethanol: Effect of nutrients.

Fermentation efficiency of more than 85% was obtained by high gravity fermentation of 33-34°Bx (spec. gravity ≈1.134) molasses medium with certain nutrients, instead of generally employed medium containing ≈16% (w/v) total sugar (spec. gravity ≈1.090) for ethanol fermentation in distilleries to get maximum 80-85% conversion. The fermenting yeast, Saccharomyces, has varied capabilities, depending on the species and nutrition for fermenting the high solids medium. The fermentation period was reduced to 48 h; and the byproducts obtained were less in concentration, upon supplementation with nutrients (or osmoprotectants) like soy flour/wheat bran to the medium in the existing batch fermentation technology. This has been found partly due to improved yeast cell viability during fermentation.

Improvement of ethanol production in very high gravity fermentation by horse gram (Dolichos biflorus) flour supplementation.

AIMS: To determine the effect of osmotic stress on yeast and to investigate the protective role of horse gram flour during very high gravity (VHG) ethanol fermentation. METHODS AND RESULTS: Saccharomyces cerevisiae was inoculated into high sugar (30-40%, w/v) containing medium with and without supplementation of horse gram flour. The fermentation experiments were carried out in batch mode. The effect of 4 or 6% of horse gram flour to the medium on the metabolic behaviour and viability of yeast was studied. Significant increase in ethanol yield up to 50% and dramatic decrease in glycerol production up to 100% was observed in the presence of horse gram flour. The fermentation rate was increased from 3 to 5 days with increased viable cell count. The physical and chemical factors of horse gram flour may aid in reducing the osmotic stress of high gravity fermentation of ethanol as well as enhancing ethanol yield. CONCLUSIONS: It was found that horse gram flour not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of high ethanol concentration by using VHG sugar fermentation eliminates the expensive steps in the conventional process and saves time.