Immobilization, enzymes whole cells

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] ... 

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. 

This chapter covers a number of applications, none of which is sufficiently developed to justify a whole chapter. This is not a reflection on the importance, interest, or possible impact of the technology, especially the first section in which we discuss the use of polyurethane to immobilize enzymes and cells. 

As in the case of enzymes, whole cells can be immobilized for several advantages over traditional cultivation techniques. By immobilizing the cells, process design can be simplified since cells attached to large particles or on surfaces are easily separated from product stream. This ensures continuous fermenter operation without the danger of cell washout. Immobilization can also provide conditions conducive to cell differentiation and cell-to-cell communication, thereby encouraging production of high yields of secondary metabolites. Immobilization can protect cells and thereby decrease problems related to shear forces. 

The immobilization concept was later extended and applied to living cells41 . Immobilization of whole cells rather than purified enzymes reduced the expense of separation, isolation and purification of the enzyme. Furthermore, in multistep reactions, in which several enzymes are involved, the application of immobilized cells is advantageous. Since the enzymes are in their native state their stability is enhanced. Such systems may widely be applied, which is not possible with isolated pure enzymes, and are less expensive than processes based on free intact cells 42). 

The commercial bioconversion process employs the enzyme nitrile hydratase, which catalyzes the same reaction as the chemical process (Figure 31.15). The bioconversion process was introduced using wild-type cells of Rhodococcus or Pseudomonas, which were grown under selective conditions for optimal enzyme induction and repression of unwanted side activities. These biocatalysts are now replaced with recombinant cells expressing nitrile hydratase. The process consists of growing and immobilizing the whole cell biocatalyst and then reacting them with aqueous acrylonitrile, which is fed incrementally. When the reaction is complete the biocatalyst is recovered and the acrylamide solution is used as is. The bioconversion process runs at 10°C compared to 70°C for the copper-catalyzed process, is able to convert 100 percent of the acrylonitrile fed compared to 80 percent and achieves 50 percent concentration... 

Transformations with immobilized enzymes or cells Often the stability of the biocatalyst can be increased by immobilization and many different enzymes and cells have been immobilized by a variety of different methods. The most popular method for the fixation of whole cells is entrapment or encapsulation with calcium alginate. Other natural gels e.g., carrageenan, collagen, chemically-modified natural polymers e.g., cellulose acetate and synthetic gels and polymers e.g., polyacrylamide or polyhydroxyethylmethacrylate can also be used for this type of immobilization. 

Other methods for the immobilization of whole cells are fixation on solid supports or matrices by adhesion or adsorption, e.g., on polyurethane foam, direct cross-linking e.g., by glutaraldehyde, or retention of the cells by hollow fibers. The simplest example of the last method is the separation of cells or enzymes from the assay by means of a dialysis lube which allows the exchange of the small substrate and product molecules but not of the larger components. This technique has been highly developed to provide the so-called membrane reactor for the fixation of enzymes. If charged ultrafiltration membranes are used, the cofactor can be retained in its native form316. 

Electrochemical monitoring of these compounds has definite advantages. For example, wide concentration ranges are measurable without dilution simply by scale switching and the test sample does not need to be optically clear. Many of these methods have utilized enzyme-catalyzed reactions because of the specificity of such reactions. Many reports on applications of enzyme electrodes in clinical and food analysis have been published (1,2). However, enzymes are generally expensive and unstable. Recently many methods have been developed for immobilization of whole cells... 

Biocatalysts can be immobilized using either the isolated enzymes or the whole cells. Immobilization of whole cells is an easier alternative to immobilization of isolated enzymes due to operational facility. But, at the same time, immobilized cells show lower catalytic activity compared with immobilized enzymes. 

Immobilization is the method of cultivation of microorganisms that allows a repeated use of biocatalysts (be it enzyme or whole cells), creating prerequisites for the production of valuable products in an automated continuous mode. The most considerable problem in using biocatalysts is related to mass transfer. In aerobic systems, low solubility of oxygen in carriers, especially in some gels and polymers, can decrease the effectiveness of biocatalyst action. In this respect, propionic acid bacteria, which do not require aeration, show certain advantages over aerobic cultures. At present, about eight different processes that use immobilized enzymes and cells have found industrial applications. These are mainly one-or two-step processes used in the manufacture of foods and pharmaceutical preparations (Vorobjeva et al, 1978). An essential characteristic of a biocatalyst is productivity. 

The immobilization of whole cells provides a means for the entrapment of multistep and cooperative enzyme system present in the intact cell, repetitive use and improved stabihty. This technique is also advantageous in the separation of bioproducts from cell mass in a continuous bioconversion process [114,115]. The other advantages of immobilized growing cells include (1) protection of cells against unfavourable environmental factors (2) changes in the permeability of the cells (3) reduced inhibition by substrate and product (4) reusability and (5) faster removal of end product. 

An important feature of this procedure is that addition of the enzyme to the reaction mixture during the formation of the gel minimizes enzyme deactivation. Furthermore, covalent incorporation of the enzyme into the gel provides some protection against proteases. Second, the procedure is simple and of general use and should be directly applicable to a variety of enzyme systems as well as immobilization of whole cells and organelles. Finally, the gel can be rendered susceptible to magnetic filtration by including a ferrofluid in the gel formation step. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

In another approach, the alcohol moiety, formed by an enzymatic hydrolysis of an ester, can act as a nucleophile. In their synthesis of pityol (8-37a), a pheromone of the elm bark beetle, Faber and coworkers [17] used an enzyme-triggered reaction of the diastereomeric mixture of ( )-epoxy ester 8-35 employing an immobilized enzyme preparation (Novo SP 409) or whole lyophilized cells of Rhodococcus erythro-polis NCIMB 11540 (Scheme 8.9). As an intermediate, the enantiopure alcohol 8-36 is formed via kinetic resolution as a mixture ofdiastereomers, which leads to the diastereomeric THF derivatives pityol (8-37a) and 8-37b as a separable mixture with a... 

Enzymes can be immobilized by matrix entrapment, by microencapsulation, by physical or ionic adsorption, by covalent binding to organic or inorganic polymer-carriers, or by whole cell immobilization (5 ). Particularly impressive is the great number of chemical reactions developed for the covalent binding of enzymes to inorganic carriers such as glass, to natural polymers such as cellulose or Sepharose, and to synthetic polymers such as nylon, polyacrylamide, and other vinyl polymers and... 

Membranes can be used as a matrix for immobilization of a catalyst. Four basic types of catalysts are relevant (a) enzymes and (b) whole cells for biocatalysis (c) oxides and (d) metals for nonbiological synthesis. Biocatalysts will be considered first since their immobilization in (or on) the membrane was explored much earlier. Five techniques have been studied in varying degrees. They are (1) enzyme contained in the spongy fiber matrix ... 

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