Immobilized cell catalysts operational stability

From a practical standpoint, the two most important characteristics of an immobilized cell catalyst are its activity and its operational stability. The latter parameter is usually expressed in catalyst half-life. The amount of activity, say in International Units (I.U.), would be a function of cell-to-carrier ratio. As mentioned earlier, a 50% loading ratio has been found to be optimal. 

Mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) were applied to water-soluble ketone substrates. For example, 4-hydroxy-2-butanone was reduced to (S)/(R)-l,3-butanediol, with a high yield and stereoselectivity. Intact E coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-hexanediamine and glutaraldehyde and evaluated in a batch reactor. This system produced (S)-l,3-butanediol (87% ee) with a space-time yield (STY) of 12.5 mg/h/mL catalyst or (R)-l,3-butanediol (99% ee.) with an STY of 60.3 mg/h/mL catalyst. The immobilized cells in a packed bed reactor continuously produced (R)-l,3-butanediol with a yield of 99% (about 49.5 g/L) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h. The concentration of PEI used for immobilization influenced the operational stability of immobilized cells, and the cells treated with 3% PEI showed better stability than those treated with lower PEI concentrations The immobilized E. coli biocatalyst could be used more than 30 times (for about 500 h) with no decrease in conversion [54]. 

As has been shown already on an Industrial scale, fermentation can be substituted by heterogeneous catalysts with resting microbial cells Immobilized In polymeric carriers. Repeated use of the once formed biomass, continuous process operation, and elimination of costly separation steps of product solution from biomass are obvious advantages of this new technology. Some principal aspects of a) Immobilization methodology, b) catalyst effectiveness, and c) operational stability shall be outlined In this contribution. 

Figure 11. Operational stability functions of E. coli cells immobilized in epoxy carrier for consecutive batch operation in the production of 6 APA from penicillin G. Top, experimental data bottom, model calculations for the superposition of 6 APA formation and conversion dependence catalyst deactivation reaction in the diffusion controlled regime.

Perhaps the first decision to be made in process development is the difficult decision of whether the enzymes to be used should be used in an integrated format. Such a question does not arise with conventional single biocatalytic steps but is highly important in multienzyme processes. One of the key criteria here is whether the enzymes can be operated together without compromise to any of the individual enzyme s activity or stability. An interaction matrix (see Section 10.6) can be used to assist such decision making. In cases where the cost of one or more of the enzyme(s) is not critical, it will be possible to combine in a one-pot operation. In other cases, where the cost of an individual enzyme becomes critical, then it may be necessary to separate the catalysts, such that each can operate under optimal conditions. Likewise, selection of the biocatalyst format (immobilized enzyme, whole cell, cell-free extract, soluble enzyme, or combinations thereof) in combination with the basic reactor type (packed bed, stirred tank, or combinations thereof) and biocatalyst recovery (mesh, microfiltration, ultrafiltration, or combinations thereof) will determine the structure of the process flowsheet and therefore is an early consideration in the development of any bioprocess. The criterion for selection of the final type of biocatalyst and reactor combination is primarily economic and may best be evaluated by the four metrics in common use to assess the economic feasibility of biocatalytic processes [29] ... 

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