Enzyme recycle reactor

Many procedures have been suggested to achieve efficient cofactor recycling, including enzymatic and non-enzymatic methods. However, the practical problems associated with the commercial application of coenzyme dependent biocatalysts have not yet been generally solved. Figure A8.18 illustrates the continuous production of L-amino adds in a multi-enzyme-membrane-reactor, where the enzymes together with NAD covalently bound to water soluble polyethylene glycol 20,000 (PEG-20,000-NAD) are retained by means of an ultrafiltration membrane. 

In analogy to the enzyme membrane reactors (EMRs) [8], a chemzyme membrane reactor (CMR) is used to retain a polymer-enlarged chemical catalyst of this kind. Tremendous progress could be made in the recycling of polymer-enlarged catalysts (Fig. 3.1.3) by employing different types of catalysts for both the enan-tioselective C-C bond formation and redox reactions. 

Membrane reactors allow a different option for the separation of biocatalysts from substrates and products and for retention in the reactor. Size-specific pores allow the substrate and product molecules, but not the enzyme molecules, to pass the membrane. Membrane reactors can be operated as CSTRs with dead-end filtration (Figure 5.5e) or as loop or recycle reactors (Figure 5.5f) with tangential (crossflow) filtration. 

Immobilized forms of penicillin amidases and acylases have replaced whole-cell biocatalysts for the production of 6-APA and 7-ACA as they can be reused many times, in some cases for over 1000 cycles. Another major advantage is the purity of the enzyme, lacking the /3-lactamase contaminants often present in whole cells. The productivity of these biocatalysts exceeds 2000 kg prod-uct/kg catalyst. A typical process for the production of 6-APA employs immobilized penicillin G acylase covalently attached to a macroporous resin. The process can be run in either batch or continuous modes. The pH of the reaction must be maintained at a value between 7.5 and 8 and requires continuous adjustment to compensate for the drop caused by the phenylacetic acid generated during the course of the reaction. Recycle reactors have been used, as they allow both pH control and the use of packed bed reactors containing the immobilized catalyst. The enzymatic process is cheaper, although not... 

Extensive investigations were performed to determine the potential of carbon-coated monoliths as supports for enzymes. The enzymes were adsorbed on the functionalized supports under ambient conditions, in a recycle reactor in which the liquid was recycled over the support under upflow conditions. A 50 mM phosphate buffer with pH 7 was used as a medium. The protein concentration was determined by using UV-VIS... 

The continuous enzyme membrane reactor (CMR). (1) Temperature-controlled water-bath (2) Feed tanig (3) Stirrer motor for feed tank (4) Feed pump (5) Feed inlet line to the reaction vessel (6) Reaction vessel (7) Magnetic stirring table (8) Prefilter (9) Recycle pum (10) Flowmeter (11) Membrane inlet pressure gauge (12) Hollow fiber membrane cartridge (13) Membrane outlet pressure gaug (1 Pressure adjusbneut valve (15) Retentate recycle line (16) Air bath environment (17) Pemieate (product) line (18) Permeate collection vessel (19) Electronic balance... 

The substrate specificity of acylase is very broad, and a wide range of pro-teinogenic and nonproteinogenic Af-acetyl and A-chloroacetyl amino acids are transformed by the enzyme. The enzyme membrane reactor (Figure 4) is operated continuously as a recycle reactor, and the enzyme is retained by a UF hollow-fiber membrane (MWCO 10000). 

Results on operational stability of both acylases in a recycle reactor at constant conversion1641 with reaction conditions close to intended large-scale conditions demonstrated much better stability of the Aspergillus enzyme, while renal enzyme is not stable enough for long-term operation164,651. Moreover, on the process scale achieved today the supply of renal acylase is insufficient, so that fungal acylase is used almost exclusively nowadays, especially since the price per unit is comparable. 

Use of Enzyme Membrane Reactors (EMR) the method of choice for systems with co-factor recycling or for reactions with expensive enzymes. 

Entrapment in polyacrylamide gel Active immobilized enzyme theoretical treatment of and experimental results for the use of the immobilized enzyme in a packed-bed differential recycle reactor 818... 

Some enzymes are produced more as a secondary metabolite, and specific productivity may then be an inverse function of the growth rate, i.e., nongrowth-associated production. Here, a recycling reactor may be the most suitable. A recycling reactor is similar to the continuous culture, but a device is added to return a significant fraction of the cells to the reactor. Low growth rates with high cell concentration can often be achieved in such systems. 

Like enzymes, whole cells are sometime immobilized by attachment to a surface or by entrapment within a carrier material. One motivation for this is similar to the motivation for using biomass recycle in a continuous process. The cells are grown under optimal conditions for cell growth but are used at conditions optimized for transformation of substrate. A great variety of reactor types have been proposed including packed beds, fluidized and spouted beds, and air-lift reactors. A semicommercial process for beer used an air-lift reactor to achieve reaction times of 1 day compared with 5-7 days for the normal batch process. Unfortunately, the beer suffered from a mismatched flavour profile that was attributed to mass transfer limitations. 

The final step is a hydrolyzing step with sulfatase enzymes (E.C. number 3.1.6.1), such as limpet sulfatase, Aerobacter aerogenes sulfatase, Abalone entrail sulfatase, or Helixpomatia sulfatase. This step was suggested to be carried out in a CSTR or fluidized bed reactors, with counter-current flow between the aqueous and the oil phase. A more efficient removal of the sulfate into the aqueous stream is expected to occur in this cross-flow manner. A final separation of the reacting mixture was suggested to obtain sulfur-free product and aqueous enzyme solution for recycle. 

Hence the dimension ("the order") of the reaction is different, even in the simplest case, and hence a comparison of the two rate constants has little meaning. Comparisons of rates are meaningful only if the catalysts follow the same mechanism and if the product formation can be expressed by the same rate equation. In this instance we can talk about rate enhancements of catalysts relative to another. If an uncatalysed reaction and a catalysed one occur simultaneously in a system we may determine what part of the product is made via the catalytic route and what part isn t. In enzyme catalysis and enzyme mimics one often compares the k, of the uncatalysed reaction with k2 of the catalysed reaction if the mechanisms of the two reactions are the same this may be a useful comparison. A practical yardstick of catalyst performance in industry is the space-time-yield mentioned above, that is to say the yield of kg of product per reactor volume per unit of time (e.g. kg product/m3.h), assuming that other factors such as catalyst costs, including recycling, and work-up costs remain the same. 

A key consideration in development of all multi-step bioprocesses is the type of bioreactor it may be necessary to accommodate a range of conditions including compartmentalization of the enzymes, cofactor recycle, adequate oxygen supply, variable temperature and pH requirements, and differential substrate feed rates. Examples described below include a range of different reactors, of which membrane bioreactors are clearly often particularly useful. 

However, the RTR is an efficient way to introduce a small amount of backmix into a reactor. This is particularly useful for an autocatalytic reactor where the rate is proportional to the concentration of a product. In the enzyme-catalyzed reactions discussed previously and modeled as A B, r = kCACs, need to seed the reactor with enzyme, and this is easily accomplished using recycle of a small amount of product back into the feed. 

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