Enzymes recovery process

FIGURE 6.5 Simplified recovery schematic for a clarified enzyme recovery process. 

Activated enzyme [6]. In an enzyme recovery process, engineers were able to model the recovery of protein [/ (f) %] and the enzyme activity [a(f) Ul/mg] as a function of time with the following equations ... 

The main factors influencing the design of the enzyme recovery process are as follows ... 

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... 

ZENECA has developed a non-solvent based recovery process as an alternative to solvent extraction for the commercial production of poly(3HB) and poly(3HB-co-3V) by A. eutrophus [94,95], In this process the cells were first exposed to a temperature of 80 °C and subsequently treated with a cocktail of various hydrolytic enzymes consisting of lysozyme, phospholipase, lecithinase, the proteinase alcalase, and others. Most of the cellular components were hy-... 

In order to develop a host strain for production of a variety of subtilisin enzymes, it was first necessary to delete the endogenous alkaline and neutral proteases (72,76). The strain was then constmcted to contain the optimal combination of subtilisin regulatory genes which were compatible with the proposed fermentation and recovery process. Once this strain had been produced, the recombinant enzyme... 

Polysaccharide degrading enzymes have a long history of commercial application in food processing, horticulture, agriculture, and protein research. As with most other industrial enzymes, the economic use of polysaccharidases often depends on obtaining the maximum activity lifetime in the process environment and/or securing a recovery system that permits the sensible reuse of active enzymes from process streams. 

The fermentation step to produce penicillin GA is the major cost element in the overall process to produce 6-APA. This is substantially due to the high cost of sterile engineering (Table 4.6 and 4.7). Clarification, extraction and solvent recovery steps are also significant, a reflection of the dilute and impure composition of fermentation broths. The concentration of 6-APA in the final broth has a big effect on total process costs. Thus increasing final 6-APA concentrations from 1.2-6.0% have been calculated to reduce production costs by over 50% (Table 4.8). By contrast the 6-APA production step cost is quite small, and is less that half the cost of the solvent recovery process (Table 4.6). The costs of the immobilized enzyme is not insignificant in a recent calculation it was estimated at 2.5 /kg 6-APA (Rasor and Tischer, 1998). 

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. 

Purified COX-2 (0.79 nmol) is treated with 1.0 mol equivalent of inhibitor and the mixture is incubated for 60 min at room temperature. The remaining activity at this time is 4% that of a vehicle-treated control. The sample is then divided in two and the protein denaturated by treatment with four volumes of ethyl acetate/methanol/1 M citric acid (30 4 1). After extraction and centrifugation (10000 g for 5 min), the organic layer is removed and the extraction repeated. The two organic layers are combined and dried under N2. The extract is dissolved in 10 pi of HPLC solvent mixture consisting of water/acetonitrile/acetic acid (50 41 0.1) and 50 pi are injected onto a Novapak C-18 column (3.9 x 150 mm) and developed at 1 ml/min. The inhibitor is detected by absorption at 260 nm and eluted with a retention time of 6.6 min in this system. Control experiments for inhibitor recovery are performed with incubation of the inhibitor in the absence of enzyme and processing of the samples in an identical fashion before quantitation by HPLC. 

Figure 30.6 shows a typical recovery process for antibiotics, and a schematic overview of a typical downstream process in an enzyme plant is given in Figure 30.7. From these diagrams it is apparent that most recovery...

As may be expected, criteria for the study of pseudo irreversible inhibitors are very similar to those for both affinity labels and mechanism-based inhibitors. However, because of the inherent reversibility of pseudoirrevers-ible inhibitors, it may be more difficult to obtain structural evidence for the covalent enzyme inhibitor adduct. Further, determination of the rate of reactivation and characterization of the products of the recovery process will also be of major importance in designating an inhibitor as pseudoirreversible. 

DESCRIPTION OF PILOT SCALE ENZYME PRODUCTION AND RECOVERY PROCESS... 

The enzymatic resolution of (R,S)-2-ethoxycarbonyl-3,6-dihydropyran has been carried out repeatedly in a pilot plant on a 100-125 kg scale. The conditions of pH, agitation, concentration, and enzyme/substrate ratio have been further optimized, but for the most part conditions developed during the laboratory optimization studies have been found to work well. This technology is currently being used to produce ton quantities of (S)-4. It has to be pointed out that no major difficulties were encountered during the scale-up. The work-up of the product in this process, unlike in many other enzyme-catalyzed processes, is quite simple and volume efficiencies are better than some chemical reactions. The recovery and recycling of the enzyme is not needed since it is commercially available and relatively inexpensive. 

Kroner, K. H., Hustedt, H., and Kula, M. R., Extractive Enzyme Recovery Economic Considerations, Process Biochemistry, 19 170-179 (Oct. 

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). 

Fruit juice clarification by pectinases and cellulases is another interesting application. In the conventional process after the enzymatic reaction in the pulp treatment step takes place, filtration over diatomaceous earth follows. This filtration-type process produces a lot of solid waste, and results in costly enzyme loss. MBR are appropriate for such application either for enzyme recovery and recycle or in the form of a more compact CMR type system, with the biocatalyst immobilized on the membrane itself [4.58]. 

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