Industrial applications of immobilized enzymes

Because enzymes can be intracellulady 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 applications of immobilized enzymes is based. 

I. Chibata and T. Tosa Industrial applications of immobilized enzymes and immobilized microbial cells. Applied Biochemistry and Bioengineering 1 (1976) 329-357. 

In 1969, we [1, 2] succeeded in the industrial application of immobilized enzyme, l.e. immobilized amlnoacylase, for the continuous production of L-amino acids from acetyl-DL-amino acids. This is the first industrial application of immobilized enzymes in the world. Since then we [3, 4, 5, 6, T also carried out the Industrial applications of Immobilized microbial cells for the continuous productions of L-aspartic acid and L-malic acid using immobilized microbial cells with polyacrylamide gel. 

In the 1970s, industrial applications of immobilized enzymes spread rapidly, contemporaneously with the development of a new method using immobilized microbial bacteria treated with acetone or heat and involving the use of an enzyme in the cell. 

The first commercial use of immobilized enzymes was developed in the mid-1960s, when amino acylase was used to separate racemates of L-amino acids by adsorption. Next came penicillin amadase for the production of semisynthetic penicillins. A major industrial application of immobilized enzymes was achieved in 1976 with the use of glucose isomerase to convert glucose to fructose. The first product was Sweetzyme developed by Novo Nordisk. Glucose was available as a by-product from the manufacture of cornstarch, but was not used as a sweetener because of its relative lack of sweetness (93). Because fructose tastes more than... 

Many novel derivatives of polysaccharides and glycoproteins, etc., prepared over the past year are reported in Chapter 8. Theoretical aspects of immobilized enzymes are also considered in some detail in this chapter. It is encouraging to note that industrial applications of immobilized enzymes have gained impetus during the past year or so. 

When immobilized glucose isomerase was introduced in the early seventies, it was believed, that other industrial applications of immobihzed enzymes would soon be found, but this turned out not to be trae. The main limitation to the use of immobilized enzymes is that the substrate has to be soluble and highly purified in order to avoid clogging of the enzyme bed. In the case of glucose isomerase the cost of purifying the syrup (carbon treatment, ion exchange) before the fixed bed isomerase reactors is substantially higher than the enzyme costs. 

Tosa, T. and Shibatani, T. (1995) Industrial Applications of Immobilized Biocatalysts in Japan. Enzyme Engineering XU, edited by M.-D.Legoy and D.N.Thomas. Aimals of the New York Academy of Science, Vol. 750, 364-375. 

W. II. Pitcher Design and Operation of Immobilized Enzyme Reactors. - S. A Barker Biotechnology of Immobilized Multienzyme Systems. - R. A Messing Carriers for Immobilized Biologically Active Systems. -P. Brodelius Industrial Applications of Immobilized Biocatalysts. - B. Solomon Starch Hydrolysis by Immobilized Enzymers. 

Swaisgood has reviewed applications of immobilized enzymes in the food industry during the past 40 yr. He discusses not only the HFCS application, but also a variety of others, some of which are no longer employed commercially. Table 3 contains a summary of these applications. 

Table 3 Applications of immobilized enzymes in the food industry...

For example see (a) Mosbach K (ed) (1987/88) Methods in enzymology immobilized enzymes and cells, parts B-D, vols 135-137. Academic Press, San Diego (b) Tanaka A, TosaT, Kobayashi (eds) (1993) Industrial application of immobilized biocatalysts, vol 16. Marcel Decker, New York (c) Kennedy JF, Melo EHMJumelK (1990) Chem Eng Prog 7 81 ... 

A range of excellent and recent reviews can be found, in which the use of enzymes within specific branches or disciplines of organic chemistry is highlighted. These include biocatalysis in carbohydrate chemistry [39], polymer chemistry [40] and for protecting group manipulations [41]. The present chapter is focused on immobilized enzymes. Hence, as an appetizer, a few selected applications with Novozym 435 are presented below, followed by a short subsection discussing industrial-scale applications of immobilized enzymes. 

Immobilization can be achieved by adsorption or covalent fixation of the biocatalyst to a solid support (e.g. surface-modified polymer or glass beads), by entrapment or by encapsulation in gel beads (e.g., agarose, polyacrylamide, alginate, etc.). Hundreds of immobilization methods have been described and reviewed in the literature [83-89], but only a limited set of methods has found real technical applications. The first large-scale applications of immobilized enzymes were established for the enantioseparation of D- and L-amino acids by Chib-ata, Tosa and co-workers at Tanabe Seiyaku Company. The Japanese achievements in the large-scale application of immobilized systems are very well documented in an excellent multi-author publication edited by Tanaka, Tosa and Kobayashi [90] (see also section 7). Some enzyme suppliers sell important industrial enzymes not only in the free form (solution or powder) but also immobilized on solid supports. 

One of the approaches to prepare more superior catalysts for application purpose is immobilization of enzymes. Over the past 10 years, the immobilization of enzyme has been the subject of increased Interest, and many papers on potential applications of immobilized enzymes and microbial cells have been published. However, practical industrial systems using immobilized enzymes and immobilized microbial cells have been very limited. 

Clearly, most biocatalytic reactions for the production of fine chemicals are used to obtain enantiopure or enantioenriched compounds, and only a minor number of syntheses lead to products without chiral centers. More than 65 applications of immobilized enzymes or whole cells for industrial research and production have been treated in this review, and it can be stated that approximately 80% utilize the class of hydrolytic enzymes. This number reflects the ease of handling and the broad utility of these enzymes. The reported hydrolytic enzyme applications mainly involve lipases, whereas other hydrolases can only be found in fewer but nevertheless just as attractive cases. The broad field of asymmetric synthesis (e.g., asymmetric reduction/oxidation) is defi-... 

Penicillin acylase is an extremely important enzyme for the industrial production of 6-aminopenicillanic acid and 7-amino 3-desacetoxicephalosporanic, as key intermediates of semi-synthetic (3-lactam antibiotics (Parmar et al. 2000). These precursors are now industrially produced mainly by hydrolysis of penicillin G and cephalosporin G with immobilized penicillin acylase, which have replaced the former cumbersome chemical processes almost completely (Bruggink 2001 Kallenberg et al. 2005), representing one of the most successful cases of industrial application of hydrolytic enzymes in bioprocesses. 

Industrial application of immobilized biocatalysts. Marcel Dekker, New York, pp 15-24 Schaffeld G, Bruzzone P, Illanes A et al. (1989) Enzymatic treatment of stickwater from fishmeal industry with the protease from Cucurbita ficifolia. Biotechnol Lett ll(7) 521-522 Schmedding DIM, van Gestel MJMC (2002) Enzymes in brewing. In Whitehurst RJ, Law BA (eds). Enzymes in food technology. CRC Press, Boca Raton, USA, pp 57-75 Schmid A, Dordick IS, Hauer B et al. (2(X)1) Industrial biocatalysis today and tomorrow. Nature 409 258-268... 

Adsorption is based on the interaction of secondary bonds on the surface of the carrier and enzyme. The first industrial-scale application of immobilized enzyme is the aminoacylase adsorbed to DEAE-Sephadex A25. Adsorption is the oldest, and easiest, economical immobihzation method. According to the different characteristics of adsorption, it can be divided into physical adsorption and ion exchange adsorption. 

Several industrial applications of biocatalysts (enzymes and cells) are listed in Table 20.11, many of which are used in immobilized form. Although in some cases the enzymatic process involved is a single-step process that gives the final product directly from the basic reactant, in most industrial processes this constitutes one step (sometimes more) in a multistep chemical synthesis, but cuts down the total number of steps and thus the cost of production. It also imparts greater selectivity and purity to the product. 

An edited series of papers on the subject of immobilized enzymes for industrial reactors cover the following aspects basic enzymology carriers including controlled-pore glasses for immobilization immobilization by covalent attachment, entrapment, adsorption, and inorganic brid formation comparative characteristics of free and immobilized enzymes inunobilized co-enzjrmes design and operation of immobilized enzyme reactors and applications of immobilized enzymes. ... 

Table 2. Possibilities for the Application of Immobilized Enzymes in Food Industry (Hartmeier, 1977) ...

Immobilized enzymes and whole cells have found well-documented applications in industry, medicine, and analytical chemistry. Theoretically, it should be possible to carry out any enzymatic reaction with the help of the respective immobilized enzyme or whole cell containing the enzyme. The technique of using an immobilized enzyme for a chemical transformation is not basically different from using the soluble enzymes. In commercial applications, the immobilized enzymes can be used in a continuous-flow reactor. However, the optimum conditions for a specific reaction will have to be redetermined before maximum turn-over can be achieved. Thus, proteolytic enzymes such as trypsin, when immobilized on an anionic matrix such as cofpolyethylene-maleic anhydride), require a much lower pH for reaction than in solution. Some typical applications of immobilized enzymes that are currently being made, or are in the process of development, are mentioned in Table 15-1. 

Until recently, these drawbacks limited the broader application of immobilized enzymes on industrial scales. Nowadays, large-scale production processes (10 -10 tons per year) using immobilized enzymes are practiced with a small number of enzymes such as lipase [10-12], lactase [13], penicillin G acylase [14], aspartase [15], and glucose isomerase [16], among others. DiCosimo and coworkers have recendy published an interesting review article on the industrial use of immobilized enzymes [5]. 

P.B. Poulsen, European and American trends in the industrial application of immobilized biocatalysts, Enzym. Microbial Technol. 3 (1981) 271-273. 

Stabilization of activated oxidoreductases on time scales of months to years has historically been challenging, and the lack of success in this regard has limited the industrial implementation of redox enzymes to applications that do not require long lifetimes. However, as mentioned in the Introduction, some possibility of improved stability has arisen from immobilization of enzymes in hydrophilic cages formed by silica sol—gels and aerogels, primarily for sensor applications.The tradeoff of this approach is expected to be a lowering of current density because... 

---