http://wolfson.huji.ac.il/expression/local/bacteria/studierAutoInduced.pdf

Protein production by auto-induction in high-density shaking cultures

https://www.massey.ac.nz/%7Eychisti/Lipases.pdf

Production, purification, characterization, and applications of lipases.

Lipases for biotechnology.

To succeed, many cells must alternate between life-styles that permit rapid growth in the presence of abundant nutrients and ones that enhance survival in the absence of those nutrients. One such change in life-style, the “acetate switch,” occurs as cells deplete their environment of acetate-producing carbon sources and begin to rely on their ability to scavenge for acetate. This review explains why, when, and how cells excrete or dissimilate acetate. The central components of the “switch” (phosphotransacetylase [PTA], acetate kinase [ACK], and AMP-forming acetyl coenzyme A synthetase [AMP-ACS]) and the behavior of cells that lack these components are introduced. Acetyl phosphate (acetyl∼P), the high-energy intermediate of acetate dissimilation, is discussed, and conditions that influence its intracellular concentration are described. Evidence is provided that acetyl∼P influences cellular processes from organelle biogenesis to cell cycle regulation and from biofilm development to pathogenesis. The merits of each mechanism proposed to explain the interaction of acetyl∼P with two-component signal transduction pathways are addressed. A short list of enzymes that generate acetyl∼P by PTA-ACKA-independent mechanisms is introduced and discussed briefly. Attention is then directed to the mechanisms used by cells to “flip the switch,” the induction and activation of the acetate-scavenging AMP-ACS. First, evidence is presented that nucleoid proteins orchestrate a progression of distinct nucleoprotein complexes to ensure proper transcription of its gene. Next, the way in which cells regulate AMP-ACS activity through reversible acetylation is described. Finally, the “acetate switch” as it exists in selected eubacteria, archaea, and eukaryotes, including humans, is described.

The acetate switch.

Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.

Bacterial lipases: an overview of production, purification and biochemical properties.

A simple and rapid colorimetric method was developed to determine the lipase activity for fat splitting Free fatty acids produced by lipase from triacylglycerols were determined by observing the color developed using cupric acetate-pyridine as a color developing reagent This modified method requires only a few minutes to determine the free fatty acids, whereas it takes over 20 min by the conventional methods which require solvent evaporation and centrifugation steps The sensitivity and reproducibility of the method were good for caproic, caprylic, capric, lauric, myristic, palmitic, stearic and oleic acids

A simple and rapid colorimetric method for determination of free fatty acids for lipase assay

Candida rugosa lipase solubilized in organic solvents in the presence of both surfactant and water could catalyze the hydrolysis of triglycerides, and kinetic analysis of the lipase-catalyzed reaction was found to be possible in this system. Among eight organic solvents tested, isooctane was most effective for the hydrolysis of olive oil in reversed micelles. Temperature effect, pH profile, K(m,app) and V(max,app) were determined. Among various chemical compounds, Cu(2+), Hg(2+), and Fe(3+) inhibited lipase severely. But the enzyme activity was restorable partially by adding histidine or glycine to the system containing these metal ions. The enzyme activity was dependent on R (molar ratio of water to surfactant) and maximum activity was obtained at R = 10.5. Upon addition of glycerol to the reversed micelles, lipase activity was affected in a different fashion depending on the R values. Stability of the lipase in reversed micelles was also dependent on R, and it was most stable at R = 5.5.

Characteristics of lipase‐catalyzed hydrolysis of olive oil in AOT–isooctane reversed micelles

In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival.

We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure D- or L-lactate. Several pta mutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to D-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in ppc made the mutant produce D-lactate like a homofermentative lactic acid bacterium. When the pta ppc double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of D-lactate production, more than 62.2 g of D-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous L-lactate pathway, an L-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain which lacked phosphotransacetylase and D-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to L-lactate as the major fermentation product, and up to 45 g of L-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of D-lactate, an indigenous fermentation product, or to the production of L-lactate, a nonindigenous fermentation product.

Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1.

The superoxide dismutase (SOD)-like activity of natural antioxidants was evaluated by measuring the inhibition of pyrogallol autoxidation that is catalyzed by the superoxide radical. Among 22 water-soluble antioxidants tested, L-ascrobic acid, L-ascorbic acid 6-palmitate, glutathione (reduced form), (+)-catechin, and (-)-epicatechin showed effective SOD-like activity. To analyze lipophilic antioxidants, an optically clear organic system composed of diethyl ether, surfactant (dioctyl sulfosuccinate, AOT) and water, called reverse micelles, was developed. The optimum concentrations of AOT, water and pyrogallol for determining SOD-like activity were found to be 50 mM, 1.3 M, and 40 mM, respectively. After proving that pyrogallol autoxidation was mediated by the superoxide anion in that system, the SOD-like activity of 24 lipophilic antioxidants was measured. Cinnamon oil, gamma-oryzanol, extract of rosemary leaf, L-alpha-lecithin, and L-alpha-cephalin exhibited activity, although the activity of some antioxidants could not be measured because of the intense color or low solubility.

Joon Shick Rhee

Papers

A simple and rapid colorimetric method for determination of free fatty acids for lipase assay

Characteristics of lipase‐catalyzed hydrolysis of olive oil in AOT–isooctane reversed micelles

Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival.

Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1.

Measurement of Superoxide Dismutase-like Activity of Natural Antioxidants