Scale biocatalyst yield

Several experiments using different organic solvents in different biphasic media are necessary to find the adequate distribution of the reaction components. A series of experiments are essential for the choice of a process and for scaling-up. Experiments using Lewis cells [44] may yield useful results for understanding equilibrium, kinetics, and interactions between organic solvent-substrate and/or organic solvent-biocatalyst. A study of two-liquid phase biotransformation systems is detailed below in Sections II-IX. 

The one-pot dynamic kinetic resolution (DKR) of ( )-l-phenylethanol lipase esterification in the presence of zeolite beta followed by saponification leads to (R)-l phenylethanol in 70 % isolated yield at a multi-gram scale. The DKR consists of two parallel reactions kinetic resolution by transesterification with an immobilized biocatalyst (lipase B from Candida antarctica) and in situ racemization over a zeolite beta (Si/Al = 150). With vinyl octanoate as the acyl donor, the desired ester of (R)-l-phenylethanol was obtained with a yield of 80 % and an ee of 98 %. The chiral secondary alcohol can be regenerated from the ester without loss of optical purity. The advantages of this method are that it uses a single liquid phase and both catalysts are solids which can be easily removed by filtration. This makes the method suitable for scale-up. The examples given here describe the multi-gram synthesis of (R)-l-phenylethyl octanoate and the hydrolysis of the ester to obtain pure (R)-l-phenylethanol. 

Enantioselective enzymatic amide hydrolyses can also be applied for the preparation of optically active organosilicon compounds. The first example of this is the kinetic resolution of the racemic [l-(phenylacetamido)ethyl] silane rac-84 using immobilized penicillin G acylase (PGA E.C. 3.5.1.11) from Escherichia coli as the biocatalyst (Scheme 18)69. (R)-selective hydrolysis of rac-84 yielded the corresponding (l-aminoethyl)silane (R)-85 which was obtained on a preparative scale in 40% yield (relative to rac-84). The enantiomeric purity of the biotransformation product was 92% ee. This method has not yet been used for the synthesis of optically active silicon compounds with the silicon atom as the center of chirality. 

The threshold value for sufficient biocatalyst stability depends on the application, as does the value for product yield. For any application in synthesis the TTN should exceed 10 000, and for large-scale processing a value of > 1 000 000 is preferred. 

The influence of eutectic media on the kinetics and productivity of biocatalysts has yet to be fully elucidated. Syntheses in eutectic suspensions have been scaled up to the pilot scale in a rotating drum reactor. The bioactive peptide Na-Cbz-L-Lys(Ne-Cbz)-Gly-L-Asp(OAll)-L-Glu(OAll)OEt was synthesized via a sequential N-to-C strategy in a heterogeneous solid-liquid mixture of the substrates in the presence of chymopapain and subtilisin as well as 16-20% (w/w) water and ethanol (Gill, 2002). At substrate concentrations of around 1 m, yields of 67-74% per step at product concentrations of 0.36, 0.49, and 0.48 kg kg-1 were achieved. The corresponding space-time yields were between 0.30 and 0.64 kg (kg d)-1 and biocatalyst reuse provided productivities of 166-312 kg product (kg enzyme)-1. 

The behavior of the recombinant biocatalyst on a larger scale is of considerable economic interest. For this reason D-methionine production was analyzed from 300mM D,L-methylthioethylhydantoin (52.3g/1), in a reaction volume 300 times that used at the laboratory scale (300ml). When system 1 was induced in optimal conditions the product yield of D-methionine reached 100% in 6h and there was no D-carbamoyl-methionine accumulation (Figure 12.15a). However, when the... 

All these compounds and many others cited in the technical and patent literature [7-23] can be obtained in variable yields and purity. What makes the method attractive is that many structurally diverse compounds can be prepared with a single strategy and one biocatalyst. The method is used for the large scale preparation of the most common commercial PL like PS, PG, and PE with mixed acyl chains. One of the general strategies to industrial compounds with defined acyl chains, as already mentioned, is to prepare glycerophosphoryl-choline (GPC) by hydrolysis of natural PC (path e. Scheme 2), chemical reacylation and exchange of the polar head via PLD catalyzed transesterification. 

Compared to batch processes, continuous processes often show a higher space-time yield. Reaction conditions may be kept within certain limits more easily. For easier scale-up of some enzyme-catalyzed reactions, the Enzyme Membrane Reactor (EMR) has been developed. The principle is shown in Fig. 7-26 A. The difference in size between a biocatalyst and the reactants enables continuous homogeneous catalysis to be achieved while retaining the catalyst in the vessel. For this purpose, commercially available ultrafiltration membranes are used. When continuously operated, the EMR behaves as a continuous stirred tank reactor (CSTR) with complete backmixing. For large-scale membrane reactors, hollow-fiber membranes or stacked flat membranes are used 129. To prevent concentration polarization on the membrane, the reaction mixture is circulated along the membrane surface by a low-shear recirculation pump (Fig. 7-26 B). 

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