Membrane bioreactors enzyme development

A liquid membrane bioreactor was developed as a means of encapsulation for a multi-enzyme system incorporating an oxidation and carbohydrate cleavage, demonstrated using a-glucosidase and glucose oxidase in the conversion of maltose to gluconic acid ... 

In the development of cell or enzyme-based processes, many process configurations exist, including batch, fed batch and continuous operation. In general, the conversion and the separation processes (downstream processing) are regarded as separate units, and most industrial processes are based on this approach. In the last decades, however, more attention is paid to the integration of conversion and separation, leading to the development of membrane bioreactors [49, 50], and some of these concepts have reached an industrial scale. The membranes used for this type of reactors are almost exclusively polymeric, as temperatures seldomly exceed 100 °C for obvious reasons. 

Generally, a distinction can be made between membrane bioreactors based on cells performing a desired conversion and processes based on enzymes. In ceU-based processes, bacteria, plant and mammalian cells are used for the production of (fine) chemicals, pharmaceuticals and food additives or for the treatment of waste streams. Enzyme-based membrane bioreactors are typically used for the degradation of natural polymeric materials Hke starch, cellulose or proteins or for the resolution of optically active components in the pharmaceutical, agrochemical, food and chemical industry [50, 51]. In general, only ultrafiltration (UF) or microfiltration (MF)-based processes have been reported and little is known on the application of reverse osmosis (RO) or nanofiltration (NF) in membrane bioreactors. Additionally, membrane contactor systems have been developed, based on micro-porous polyolefin or teflon membranes [52-55]. 

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. 

Membrane bioreactors have been reported for the production of diltiazem chiral intermediate with a multiphase/extractive enzyme membrane reactor [15, 16]. The reaction was carried out in a two-separate phase reactor. Here, the membrane had the double role of confining the enzyme and keeping the two phases in contact while maintaining them in two different compartments. This is the case of the multiphase/ extractive membrane reactor developed on a productive scale for the production of a chiral intermediate of diltiazem ((2R,3S)-methylmethoxyphenylglycidate), a drug used in the treatment of hypertension and angina [15]. The principle is illustrated in... 

Ability of WSP to interact with enzymes, drugs, selective and chelating properties of many polyelectrolytes make them very perspective in the development of liquid membrane bioreactors. It is known [103] that formation of a ternary metal ion-carrier-chelator complex at the inner vesicle waU can enhance the overaU selectivity in accordance with a multiplicative, rather than additive, function of equihbrium metal-hgand binding constants. Enzyme-containing lipid vesicles (liposomes), which are hpid dispersions that contain water-soluble enzymes in the trapped aqueous space, may be named as hquid membrane micro- or nanobioreactors with intraped WSP. Preparation and properties of hpid vesicles are described in [104] review. 

Steady state models of membrane bioreactors utilizing a multi-enzyme system, which in addition to the main reaction promotes the simultaneous regeneration of the co-factor (for further discussion see Chapter 4) have been developed by different groups in Japan [5.109, 5.110]. Several of these studies have also considered the effect of backmixing [5.111, 5.112]. A model of an enzymatic hollow fiber membrane bioreactor with a single enzyme, which utilizes two different substrates (reaction 5.42) has been developed recently by Salzman et al [5.113]. 

For biotechnological applications, synthetic membranes entrapping enzymes, bacteria, or animal cells are used in membrane bioreactors disclosing new important developments mainly due to the increased stability of immobilized enzymes, the possibility of their continuous reuse and the absence of pollution of the products. Membrane bioreactors are of great interest as well for the possibility of continuously removing metabolites whose presence in the reaction environment could reduce the productivity of the reactor. 

Enzymes can convert lignocellulosic biomass into a suitable fermentation feed-stock for biofuel production. Different yeast strains are used for ethanol production, such as S. diastaticus, Candida sp., S. cerevisiae and K. marxianus, as well as different bacteria such as Zymomonas mobilis. The employment of distillation is desirable for food grade purity of applications other than that of biofuel. In fact, batch fermentation was coupled with a membrane distillation process developed with the application of a membrane distillation bioreactor for ethanol production. Meanwhile,... 

Other bioreactor configurations have been developed specifically for immobilized enzymes and cells. Enzymes immobilized within polymeric membranes are used in hollow fiber (Fig. 16) and spiral membrane bioreactors (Fig. 17). In the hollow fiber device, many fibers are held in a shell-and-tube configuration (Fig. 16) and the reactant solution (or feed) flows inside the hollow fibers. The permeate that has passed through the porous walls of the fibers is collected on the shell side and contains the product of the enzymatic reaction. Also, instead of being immobilized in the fiber wall, enzymes bound to a soluble inert polymer may be held in solution that flows inside the hollow fiber. The soluble product of the reaction then passes through the fiber wall and is collected on the shell side the enzyme molecule, sometimes linked to a soluble polymer, is too large to pass through the fiber wall. 

Identifying an environment that avoids induction of undesired enzymes and repression of desired ones and implementing bioreactor control systems that maintain these desired conditions in a bioprocess are subjects of future importance. For example, accumulation of a product in the cell environment can often repress synthesis of some of the enzymes required for production of that compound. Product repression and inhibition phenomena have motivated special interest recently in combined bioprocessing operations which accomplish separation simultaneously with bioreaction. By continuously removing a product that inhibits its own synthesis, production of that material is improved. Development of new selective membranes and other process strategies for accomplishing these separations is an important area for future research. 

The rising need for new separation processes for the biotechnology industry and the increasing attention towards development of new industrial eruyme processes demonstrate a potential for the use of liquid membranes (LMs). This technique is particularly appropriate for multiple enzyme / cofactor systems since any number of enzymes as well as other molecules can be coencapsulated. This paper focuses on the application of LMs for enzyme encapsulation. The formulation and properties of LMs are first introduced for those unfamiliar with the technique. Special attention is paid to carrier-facilitated transport of amino acids in LMs, since this is a central feature involved in the operation of many LM encapsulated enzyme bioreactor systems. Current work in this laboratory with a tyrosinase/ ascorbate system for isolation of reactive intermediate oxidation products related to L-DOPA is discussed. A brief review of previous LM enzyme systems and reactor configurations is included for reference. 

The development of a continuous system, in which immobilized enzymes can be used and both solvents can be recycled, would decrease overall costs. Hernandez et al. (2006) and de los Rios et al. (2007b) developed a process combining an IL-SC-CO2 system with membrane technology to synthesize vinyl propionate from vinyl propionate and 1-butanol at 50°C and 80 bar in a recirculating bioreactor with the presence of coated immobilized C. antarctica lipase B within the IL. It was found that the selectivity of the lipase increased with the use of ILs, compared to that achieved with supercritical carbon dioxide tested alone. 

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