Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Partial purification and characterization of extracellular protease from a halophilic and thermotolerant strain Streptomyces pseudogrisiolus NRC-15.

An alkaline protease was purified from a halophilic and thermotolerant potent alkaline protease-producing strain Streptomyces pseudogrisiolus NRC-15 using ammonium sulphate precipitation and Sephadex G-100 column chromatography. The enzyme was purified to 77.24-folds with a yield of 91.8% and the specific activity was 112 U/mg of protein. The protease showed a single band on SDS-PAGE with its molecular mass at 20 kDa and exhibited a maximum relative activity of 100% using casein as a substrate and. The enzyme had an optimum pH of 9.5 and displayed optimum activity at 50°C. The enzyme activity was completely inhibited by the serine protease inhibitor PMSF, suggesting the presence of serine residue in the active site. The enzyme activity was increased by the metal ions Ca2+, Co2+, K+ and Mg2+. The enzyme significantly enhanced the removal of stains when used with wheel detergent, indicating the potential of the enzyme for using as a laundry detergent additive to improve the performance of heavy-duty laundry detergent.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Moderate exercise training promotes adaptations in coronary blood flow and adenosine production in normotensive rats

OBJECTIVES: Aerobic exercise training prevents cardiovascular risks. Regular exercise promotes functional and structural adaptations that are associated with several cardiovascular benefits. The aim of this study is to investigate the effects of swimming training on coronary blood flow, adenosine production and cardiac capillaries in normotensive rats. METHODS: Wistar rats were randomly divided into two groups: control (C) and trained (T). An exercise protocol was performed for 10 weeks and 60 min/day with a tail overload of 5 percent bodyweight. Coronary blood flow was quantified with a color microsphere technique, and cardiac capillaries were quantified using light microscopy. Adenine nucleotide hydrolysis was evaluated by enzymatic activity, and protein expression was evaluated by western blot. The results are presented as the means ± SEMs (p<0.05). RESULTS: Exercise training increased the coronary blood flow and the myocardial capillary-to-fiber ratio. Moreover, the circulating and cardiac extracellular adenine nucleotide hydrolysis was higher in the trained rats than in the sedentary rats due to the increased activity and protein expression of enzymes, such as E-NTPDase and 59- nucleotidase. CONCLUSIONS: Swimming training increases coronary blood flow, number of cardiac capillaries, and adenine nucleotide hydrolysis. Increased adenosine production may be an important contributor to the enhanced coronary blood flow and angiogenesis that were observed in the exercise-trained rats; collectively, these results suggest improved myocardial perfusion.

New Directions in Inflammation and Immunity: The Multi-functional Role of the Extracellular RNA/RNase System.

In the mid-eighties of the last century, extracellular-proteolipid complexes have been identified in tumor patients and circulating RNA was suggested to represent a specific secretory product of cancer cells. The presence of specific types of RNA in a variety of cancer types proved to be useful in cancer diagnosis. It has been suggested that extracellular RNA and DNA are not inert molecules, but contain biological activities. Recent data have demonstrated that extracellular RNA is likely to present the up to now undefined “natural foreign surface”, serving as an initiating factor in blood coagulation in vivo. Yet, extracellular RNA seems to have even more functions. Investigations on blood-brain-barrier have shown that extracellular RNA mediates endothelial permeability. Ample success has been achieved in administrating RNase in different animal models of vascular diseases, thereby significantly delaying thrombus formation and reducing cerebral edema formation with neuroprotection in acute stroke models. Furthermore, extracellular mammalian RNA was found to decrease tumor yield in a murine model system, suggesting that extracellular RNA might trigger immune response. Finally, extracellular nucleic acids were identified as danger signals involved in innate immunity related to neutrophil-mediated bacterial killing and haemocyte activation and coagulation in the insects. Thus, a new area of research on extracellular RNA functions with promising future perspectives just started in the field of inflammation and immunity.

Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490.

The physical parameters for the production of thermostable, alkaline lipase from Arthrobacter sp. BGCC# 490 were optimized using response surface methodology (RSM), employing face centered central composite design (FCCCD). The design was employed by selecting pH, temperature and incubation period as the model factors and to achieve maximum yield, interaction of these factors was studied by RSM. A second-order quadratic model and response surface method showed that the optimum conditions for lipase production (pH 10.0, temperature 40oC and incubation period 48 h) resulted in 1.6-fold increase in lipase production (13.75 EUml-1), as compared to the initial level (8.6 EUml-1) after 48 h of incubation, whereas its value predicted by the quadratic model was 12.8 EUml-1. Lipase showed stability in the pH range 8-10 and temperature range 40-60oC, with maximum activity at pH 9.0 and temperature 50oC. Lipase activity was enhanced in the presence of K+, Ca2+ and Mg2+ ions, but inhibited by Hg2+ ions. The enzyme exhibited high activity in the presence of acetone, isopropanol and ethanol, but was unaffected by methanol. These properties suggest that the lipase may find potential applications in the detergent industry. The present work also demonstrated the feasibility of using experimental design tools to optimize physical parameters for lipase production by an indigenous Arthrobacter sp.

Regulation of the renal proximal tubule second sodium pump by angiotensins

For several years it was believed that angiotensin II (Ang II) alone mediated the effects of the renin-angiotensin system. However, it has been observed that other peptides of this system, such as angiotensin-(1-7) (Ang-(1-7)), present biological activity. The effect of Ang II and Ang-(1-7) on renal sodium excretion has been associated, at least in part, with modulation of proximal tubule sodium reabsorption. In the present review, we discuss the evidence for the involvement of Na+-ATPase, called the second sodium pump, as a target for the actions of these compounds in the regulation of proximal tubule sodium reabsorption