Recovery , enzyme

Example 6 Enzyme Recovery Using Aqueous Extraction... 

Extraction for enzyme recovery is a common process. Polyethylene glycol-dextran mixture is used to recover a-amylase from fermentation broth. Given a partition coefficient of 4.2, calculate the maximum enzyme recovery when... 

Kroner, K. H., Hustedt, H. and Kula, M-R. Proc. Biochem. No. 10 (1984) 170-179. Extractive enzyme recovery - economic considerations. 

An important issue to be addressed in industrial applications of two-phase polymer systems for enzyme recovery is the economic or product quality requirement that the phase-forming polymers and salts be recycled. 

An amidrazone (58) derived from 5-amino-5-deoxy-L-fuconolactam was found to inhibit a recombinant human a-L-fucosidase with a K -value of 820 nmol/1 [ 111 ]. A simple synthesis of 1,5-dideoxy-1,5-imino-D-arabinitol (59), previously prepared by Ganem et al. [49] as a potential maimosidase inhibitor, was applied to the affinity purification of a-L-fucosidase from bovine kidney by an improved method and the characterization of the enzyme thus obtained [112]. The relatively low affinity of this compound to the enzyme (Kj 2.2 pmol/1 at pH 7) compared to 1-deoxyfuconoJirimycin (51) turned out to be advantageous in terms of enzyme recovery and yield. Structurally related, suitably protected 5-amino-5-deoxy-D-arabinopyranose (60), was coupled with a N-acetyl-6-deoxy-6-thio-D-glucosaminide (61) to give a stable thioglycoside (62) [113]. 

Atkinson, T., Scawen, M.D. and Hammond, P.M. (1987) Large scale Industrial Techniques of enzyme recovery. In Biotechnology 7a, edited by H.Rhem and G.Reed, p. 279. Weinheim VCH Verlag. 

Figure 12. Relation of glucose selling price to delivered cellulose substrate costs for enzymatic hydrolysis process. Data for 250,000 t/yr plant 90% overall process efficiency, 50% enzyme recovery (reuse).

Finally, in a concluding paper Dr. Jorg Thommes considers enzyme recovery in Fluidized Bed Adsorption as a Primary Recovery Step in Protein Purification . As important as it is to track down new enzymes and selectively modify them, it remains equally important to actually make them available in the flask on the bench in adequate quantities at low cost with sufficient purity. Recovery is of central significance in this respect. Fluidized bed adsorption combines the process steps of cell separation, concentration and primary cleaning in recovery work. The procedure can also be excellently transferred from the laboratory to the pilot scale. 

The enzyme has a monomer weight of 30 kDa and a Km and Vmax for L-pan-tolactone of 7 mM and 30 U mg-1, respectively. X-ray fluorescence spectroscopy of crystals, and renaturation of urea/EDTA-denatured Lph in the presence of Zn2+, Mn2+, Co2+, or Ni2+ indicated Lph to be a Zn2+-hydrolase. Kinetic resolution of rac-pantolactone proceeds similarly to the fungal process mentioned above except that L-pantolactone is hydrolyzed and D-pantolactone is left behind. Repeated batches with isolated Lph and enzyme recovery by membrane filtration give d-pantolactone with 50% yield and 90-95% ee over 6 days. 

FIGURE 4 Combined forward and back extraction involving reversed micelles. (From M. Dekker, K. Van t Riet, S. R. Weijers, J. W. Baltussen, C. Laane, and B. H. Bijisterboch, Enzyme recovery by liquid-liquid extraction using reversed micelles. Chem. Eng. J. 33, B27-33, 1986, with permission from Elsevier Science.)... 

ATP-extraction followed by backextraction showed a considerable improvement over purification with protamine sulfate precipitation. A multistage ATP extraction followed by multistage backextraction should further improve the enzyme recovery. This would be in keeping with the observations of Lee et al.66 and of Sebastiao et al.69... 

Even though efficient enzyme-recovery and enzyme-fixation systems are available, they must be used under carefully controlled conditions in order to prevent loss of enzyme activity by accidental deviation from defined temperature and pH conditions and by bacterial contamination. 

In reversible inhibition, enzymatic activity is regained by the systemic elimination of inhibitor/ such that the time to enzyme recovery is dependent on the elimination half-life of the inhibitor. Competitive inhibition is characterized by competition between substrate and inhibitor for the enzyme s active site. Competition for enzyme binding can be overcome by increasing the concentration of substrate/ thereby sustaining the... 

Immobilization, dehned as the physical confinement or localization of an enzyme into a specihc micro-environment, has been a very common approach to prepare enzymes for aqueous as well as nonaqueous applications. For nonaqueous enzymol-ogy, immobilization improves storage and thermal stability, facilitates enzyme recovery, and enhances enzyme dispersion. In addition, immobilized enzymes are readily incorporated in packed bed bioreactors, allowing for continuous operation of reactions. Moreover, lyophilized enzyme powders often aggregate and attach to reactor walls, particularly when the water activity is moderately high. The major disadvantage of immobilization is low activity, induced by pore diffusion mass transfer limitations and by alteration of protein stmcture. For enzymes in nonaqueous media, the following broad categories of immobilization exist ... 

The use of these models in predicting the release and breakdown of microbial proteins, and the application of the structured model to enzyme recovery will also be discussed. 

Figure 8 shows a simulation of enzyme recovery from the wall, cytosol and mitochondria. The concentrations of recoverable enzyme are normalized to the initial amount of enzyme present in the cell site. The curves rise as enzyme is released from a site, then fall as it is hydrolyzed. It may be seen that the lytic system is usable even as a crude preparation to recover wall linked yeast enzymes in 60 to 80% yield. The yield of yeast wall enzyme depends on... 

Figure 9 Enzyme recovery from subcellular structures...

Large scale purification of enzymes in PEG/dextran and PEG/salt have been reported and reviewed by Kula et. al.(18. 19. 20). Examples include pullulanase and 1,4-j-glucan phosphorylase <21 , a-amylase (22), b-galactosidase (jg3), and the general economics of extractive enzyme recovery (24). Process studies have covered the use of continuous centrifuges (18. 19) and countercurrent distribution trains (2j 25). 

---