Detection and partial purification of two lipases fromCandida rugosa

Summary Two distinct lipases produced byCanadida rugosa were identified and separated by a high resolution anion-exchange column (Mono Q) after an ethanol extraction of the crude lipase. From this Mono Q column, lipase I eluted at 0.05 M NaCl whereas lipase II eluted at 0.15 M NaCl. The less anionic nature of lipase I was also confirmed by native polyacrylamide gel electrophoresis as well as isoelectrophoresis. Both proteins have an apparent molecular weight of 58,000 by SDS-PAGE. The isoelectric points of lipase I and II are 5.6 and 5.8 respectively.     

Evaluation and elimination of inhibitory effects of salts and heavy metal ions on biodegradation of Congo red by Pseudomonas sp. mutant

In this study, it was attempted to evaluate the influences and also recommended some elimination methods for inhibitory effects offered by salts and heavy metal ions. Congo red dye solution treated with mutant Pseudomonas sp. was taken as a model system for study. The salts used in this study are NaCl, CaCl₂ and MgSO₄·7H₂O. Though the growth was inhibited at concentrations above 4 g/l, toleration was achieved by acclimatization process. In case of heavy metal ions, Cr (VI) showed low inhibition up to 500 mg/l of concentration, compared to Zn (II) and Cu (II). It was due to the presence of chromium reductase enzyme which was confirmed by SDS-PAGE. Zn (II) and Cu (II) ion inhibitions were eliminated by chelation with EDTA. The critical ion concentrations obtained as per Han-Levenspiel model for Cr (VI), Zn (II) and Cu (II) were 0.8958, 0.3028 and 0.204 g/l respectively.     

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.     

In vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and virulence factor production in Aeromonas hydrophila

Rosmarinic acid (RA) was assessed for its quorum sensing inhibitory (QSI) potential against Aeromonas hydrophila strains AH 1, AH 12 and MTCC 1739. The pathogenic strains of A. hydrophila were isolated from infected zebrafish and identified through biochemical analysis and amplification of a species-specific gene (rpsL). The biofilm inhibitory concentration (BIC) of RA against A. hydrophila strains was found to be 750 μg ml^?1. At this concentration, RA reduced the QS mediated hemolysin, lipase and elastase production in A. hydrophila. In FT-IR analysis, RA treated A. hydrophila cells showed a reduction in cellular components. Gene expression analysis confirmed the down-regulation of virulence genes such as ahh1, aerA, lip and ahyB. A. hydrophila infected zebrafish upon treatment with RA showed increased survival rates. Thus, the present study demonstrates the use of RA as a plausible phytotherapeutic compound to control QS mediated biofilm formation and virulence factor production in A. hydrophila.     

Upper esophageal sphincter function during gastroesophageal reflux events revisited

Upper esophageal sphincter (UES) function during gastroesophageal reflux events is not completely elucidated because previous studies addressing this issue yielded conflicting results. We reexamined the UES pressure response to intraluminal esophageal pressure and pH changes induced by reflux events. We studied 14 healthy, asymptomatic volunteers (age 49 +/- 6 yr) and 7 gastroesophageal reflux disease patients (age 48 +/- 5 yr). UES pressure, intraesophageal pressure, and pH were monitored at the distal, middle, and proximal esophagus concurrently in the supine position 1 h before and 2 h after a 1,000-calorie meal. A total of 321 reflux events were identified by the development of abrupt reflux-induced intraesophageal pressure increase (IPI); 285 events occurred in patients and 36 in control subjects. In control subjects 33 of 36 and in patients 252 of 285 IPI events were associated with a pH drop. Among patients and control subjects, 99% and 100%, respectively, of all IPI events irrespective of pH drop were associated with abrupt increase in UES pressure (34 +/- 2 and 27 +/- 6 mmHg, respectively). The average percentage of maximum UES pressure increase over prereflux values ranged between 66% and 96% (control subjects) and 34% and 122% (patients). IPIs induced by both acidic and nonacidic reflux events evoke strong UES contractile responses.     

Degradation of Tectilon Yellow 2G by hybrid technique: combination of sonolysis and biodegradation using mutant Pseudomonas putida

Degradation of Tectilon Yellow 2G (TY2G), an azo dye has been studied by hybrid technique involving pretreatment by sonochemical method and further biological treatment by Pseudomonas putida mutant. Pretreatment experiments were carried out by sonolysis of the dye solution at different concentrations (100-1000 mg/L). Wild type Gram-negative P. putida species isolated from the textile effluent contaminated soil, which was found to be effective towards dye degradation, has been acclimatized so as to consume TY2G as the sole source of nutrition. Mutant strain was obtained from the acclimatized species by random mutagenesis using the chemical mutagen ethidium bromide for various time intervals (6-30 min). The optimum mutagenesis exposure time for obtaining the most efficient species for dye degradation was found to be 18 min. An efficient mutant strain P. putida ACT 1 has been isolated and was used for growth experiments. The mutant strain showed a better growth compared to the wild strain. The substrate utilization kinetics has been modeled using Monod and Haldane model equations of which the Haldane model provided a better fit. The enzyme kinetics of the mutant and wild species was obtained using Michaelis-Menten equation. The mutated species showed better enzyme kinetics towards the degradation of TY2G.