Butanol enzyme extraction

In 1954, Beaufay and de Duve (27) first suggested a relationship between microsomal phospholipid and glucose-6-phosphatase. They observed a loss of enzymic activity from phospholipid-rich microsomal preparations concomitant with extraction with such organic solvents as butanol or treatment with lecithinase. Various studies were carried out to demonstrate that the latter effect was not produced through inhibition of enzymic activity by accumulated products of the hydrolysis of phospholipids. On the basis of their observations that deoxycholate treatment labilized microsomes to phospholipase action, they concluded that . . . the detergent did not exert its primary effect on the dissociation of phospholipids from microsomal protein, but that it probably disrupted... 

Similarly, enantiomerically pure hydrophobic (S)-l-phenyl-2-propanol, (S)-4-phe-nyl-2-butanol, and (S)-6-methylhept-5-en-2-ol (sulcatol) were obtained with high purities in a dual-loop enzyme membrane reactor unit with separate membrane extraction unit (Kruse, 1996). Whereas substrate concentrations were low at 9-12 mM, space-time yields higher than 100 g (L d) 1 as well as concentrated product solutions were obtained. 

The brown algae Ecklonia cava is known for its antioxidant and anti-inflammatory functions. The butanol extract of E. cava reduces Ap secretion from HEK293 cells expressing APP with Swedish mutation and increases soluble APPa and C-terminal fragment-a (CTFa), of which activity is similar to BACE (P-site of APP cleaving enzyme) inhibitors. The extract inhibits Ap oligomerization, particularly midsize oligomer formation, and protects primary cortical neurons from various Ap-induced cell deaths [210]. 

In this laboratory, attempts (G6, G8) have been made to purify and crystallize human placental alkaline phosphatase enzyme by a number of procedures involving homogenization with 0.05 M Tris buffer (pH 8.6), extraction with butanol, ammonium sulfate precipitation, exposure to heat, ammonium sulfate fractionation, dialysis, repeated ethanol fractionation, gel filtration with Sephadex G-200 (Fig. 18), continuous curtain electrophoresis on paper (Beckman Model CP), multiple TEAE-cellulose anion exchange chromatography, and equilibrium dialysis. Variant A (electrophoretically fast-moving) of human placental alkaline... 

Starch-gel electrophoresis of the alkaline phosphatase in the butanol extracts of leukocytes revealed three variants of the enzyme. Peacock et al. (PI) have devised a method for leukocyte alkaline phosphatase assay. An additional variant was detected in blood leukocytes of leukemia patients treated with 6-mercaptopurine (RIO). Robinson and Pierce (R7) indicated that there might be a fundamental difference in molecular structure of the human serum alkaline phosphatase proteins because serum alkaline phosphatase, when incubated with neuraminidase prior to electrophoresis, demonstrated reduced anodal migration of those isoenzymes that are not L-phenylalanine-sensitive. L-Phenylalanine-sensi-tive enzyme of intestinal origin was found to be neuraminidase-resistant. 

Harris and his group (R15, R16) in London recently initiated genetic studies on human placental alkaline phosphatase, and demonstrated phenotypic differences in this enzyme. In this study, butanol extracts of alkaline phosphatase from each of 338 placentas were prepared, and the enzyme preparations were subjected to starch-gel electrophoresis at two different pH s (8.6 and 6.0). The electrophoretic patterns obtained were classified in six different groups representing six distinct phenotypes. The... 

Fast-moving bands of serum alkaline phosphatase essentially would represent metabolically altered intestinal alkaline phosphatase (analogous to the enzyme products in aging butanol extracts Fig. 21). 

M33. Moss, D. W., Iso-enzymes of alkaline phosphatase in autolysed and butanol-extracted liver preparations. Nature 193, 981-982 (1962). 

The effect of detoxification of the medium by removal of toxic compounds with UF membranes was demonstrated by Boyaval et al. [36] in the fermentation of propionic acid. UF runs led to an eightfold increase in volumetric productivity relative to fed batch experiments. The effectiveness of membrane bioreactors in the lowering of toxicity of the compounds involved in the bioconversion system was demonstrated by Edwards and co-workers [159]. An eightfold increase in the removal of phenoHc compounds from effluents was observed when polyphenoloxidase was immobilized in a capillary poly(ether)sulfone membrane as compared to the use of the free enzyme. Butanol recovery from the fermentation medium with organic solvent extraction or membrane solvent extraction led to similar results, both processes leading to decreased product inhibition. Due to the low toxicity of the extractive solvent used (isopropyl myristate) on Clostridium beyerinckii cells, no protective effect of the membrane was observed. However, precipitates observed in two-Hquid phase extraction were not observed... 

In many cases, minced tissue is extracted with a buffer solution and then centrifuged. In some cases, membrane enzymes that are more firmly bovmd to the structural framework of mitochondria require special methods these include drying with acetone, treatment with butanol-water mixtures, extraction of dried mitochondria with various organic solvents, extraction using aqueous solutions of detergents, treatment with chaotropic ions, or exposure to the action of hydrolytic enzymes. 

Alkaline Phosphatases.—Various degrees of specific interaction between concanavalin A and human alkaline phosphatases, extracted by n-butanol from various sources, have been demonstrated by a simple electrophoretic retardation technique and affinity chromatography. The lectin-binding abilities of the enzymes demonstrated the glycoproteinaceous characters of the latter. 

It should be taken into account in lipid extraction that the phospholipase D activities are relatively high in ripe cereals and this enzyme transfers the phosphatidyl residue of phospholipids to alcohols, which are used to extract lipids (cf. 3.7.1.2.1). The enzyme is inactivated during extraction with boiling water-saturated butanol. A phospholipase that hydrolyzes both acyl residues in the lecithin molecule ( phospholipase B ) has been found in germinating cereal. It influences the foam stability in beer (cf. 20.1.7.9). 

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