Extraction of enzymes

Another method is based on the same principle,112 in which the [14C]labelled methyl ester of D-galacturonan is prepared by esterification of pectic acid with [,4C]diazomethane. In the course of the enzymic de-esterification, aliquots are removed, and the unreacted substrate is precipitated with acidified ethanol or 1-propanol. After centrifugation, the labelled methanol in the supernatant liquor is determined in a liquid scintillation counter. An advantage of this method lies in the possibility of using, as substrates, short-chain oligo-D-galactosiduronates partially esterified with [14C]methanol. These substrates, beginning with the trisaccharide, are not soluble in 1 4 80% phenol-diethyl ether, which is used for the extraction of enzymically released, labelled methanol. 

Hustedt, H., Kroner, K. H. and Papmichael, N. Proc. Biochem. (October, 1988) 129-137. Continuous cross-current aqueous 2-phase extraction of enzymes from biomass - automated recovery in production scale. 

Kroner, K. H., Hustedt, H. and Kula, M-R. Biotech. Bioeng. 24 (1982) 1015-1045. Evaluation of crude dextrain as phase-forming polymer for the extraction of enzymes in aqueous 2-phase systems in large scale. 

G. Palleschi, Extraction of enzyme inhibitors using a mixture of organic solvent and aqueous solution and their detection with electrochemical biosensors. The Eighth World Congress on Biosensors. P 3.7.64, Abstract Book. Granada, Spain, 2004. 

Roe SD (1987) Whole broth extraction of enzymes from fermentation broths using commerically available adsorbents. In Verall MS, Hudson MJ (ed) Separations for Biotechnology. Ellis Horwood, Chichester, p 210... 

In order to improve the extraction of enzymes, organic solvents and surfactants are sometimes used. 

The necessity of re-focusing experiments was demonstrated with an extract of enzyme from rat caudate nuclei. In this extract a fourth peak was sometimes obtained at pH 6.5 to 7.0. The peak disappeared on re-focusing and the enzyme activity was recovered in the normal range of pH 7.5 to 8.3. This peak is therefore probably an artifact resulting from protein-enzyme interaction. 

Crude extracts of enzymes are also obtained from macerated tissues, germination seeds, fermented bran, or the "beer" from the submerged fermentation of selected strains of microorganisms. After extraction and prefiltration to remove suspended solids and particulates, UF is applied as above. 

Kroner FIK, Kula MR (1978) Extraction of enzymes in aqueous two-phase systems. Proc Biochem 13(4) 7-9... 

Pierpoint WS (2004) The extraction of enzymes from plant tissues rich in phenolic compounds. 

These studies on model enzyme complexes imply that the organic compounds which stabilize exocellular enzymes in soil are brown-coloured humic materials, probably aromatic in character. However, knowledge of the nature of the organic ligands and of the manner in which they may complex active enzymes, rests in part on the extraction of enzymes from soil, preferably in high yield and without artefact formation, and their characterization after fractionation and purification. 

Centrifugal contacting devices have heen employed to demonstrate reverse micellar extraction of enzymes on an industrial scale (Dekker et al., 1991). Spray columns have heen employed in a number of studies to study mass transfer in such systems. The mass-transfer rate of protein lysozyme being extracted was satisfactorily described in terms of the mass-transfer coefficients of the reverse micelle (solvent) phase and the aqueous phase (Lye et al., 1996). Spray extraction columns have also been used to study the extraction of enzymes in aqueous two-phase systems (Arsalani et al., 2005). 

W. Anderson, Extraction of enzymes and subcellular organelles from plant tissues. Phytochemistry 7 1973 (1968). 

A. Tissue Slice Technique. B. Tissue Homogenates. C. Fractionation of Cellular Components. D. Methods of Extraction of Enzymes. E. Protein Fractionation. F. Preparation of Buffers. 

---