Enzyme technical applications

Biopract provides technological products and processes for industry, agriculture, and environment. They not only produce technical enzyme preparations but also develop enzymes for applications in agriculture, food, and textile industry as well as in environmental technologies. On the later, bioremediation has been an area of service delivery from Biopract. Their activities regards microbial preparations for the bioremediation of organic contaminants (mineral oil (MKW), polycyclic aromatic hydrocarbons (PAH), benzene, toluene, ethylbenzene, xylene (BTEX), methyl-tert-butyl ether (MTBE), volatile organic hydrocarbons (VOC), and dimethyl sulfoxide (DMSO)). 

The results presented here demonstrate the first successful applications of the concept of electroenzymatic synthesis. However, it is also obvious that many more systems are potentially very interesting for synthetic applications. This area is wide open for further creative research and only the tip of the iceberg has surfaced. What is especially promising is the fact that electroenzymatic synthesis is an environmentally friendly technique using the electrode as a clean reagent and the enzymes for high selectivity. Closed systems for technical applications are easier to realize than in many other areas. The author is convinced that industrial syntheses of valued small scale products will be seen in the near future. 

After a long initial period of about a hundred years, with mysterious theories and several technical applications, where one (diastase) even achieved economic importance, research on enzymes obtained a chemically scientific status. 

HNLs comprise a heterogenous enzyme family, since hydroxynitrile lyase activity has evolved in different structural frames by convergent evolution [17, 18]. Thus, (S) -specific HNLs based on an a/P-hydrolase fold framework from Manihot esculmta (cassava) [19-21], Hevea hrasilensis (rubber tree) [22-26], and Sorghum hicolor (millet) [27-33] have been described. (R)-specific HNLs based on the structural framework of oxidoreductases were isolated from Linum usitatissimum (flax) [30, 34-37] and Rosaceae (e.g., bitter almonds) [31, 38]. Despite their potential in biocatalysis only few HNLs (from cassava and rubber tree) are available by recombinant gene expression, which is a prerequisite for their technical application [20, 24]. Thus, cloning, recombinant expression, and... 

Technical Enzymes. When an enzyme is used for a technical application, ie, industrial but nonfood and nonfeed, its regulatory status is determined by its properties as a naturally occurring substance. These properties determine the classification and consequent labeling in accordance with existing schemes for chemicals. It should be noted that enzymes are not listed as dangerous chemicals. 

Bearing in mind technical application with scale-up, enzyme immobilization is a prerequisite. Since the large enzyme (ca. 180 kDa) is additionally linked with a dextran chain, it can be easily and economically immobilized by entrapment in calcium alginate [29, 94]. 

A further important aspect is the feasibility of whole cell biotransformations. Whole cell biotransformations show a lot of advantages as compared to isolated enzymes, such as the improved stability of enzymes. If both, producing and regenerating enzymes, are available in one single strain, no addition of expensive cofactor is necessary because the intracellular cofactor pool can be utilized. Whole cell biotransformations are therefore very promising for technical applications, and making these conversions an intensively studied subject in the last years. The use of recombinant DNA techniques offers many possibilities to create capable systems. This chapter describes the most important whole cell biotransformations developed in the past as well as relevant processes with small-scale and technical application. 

Wild type cells often contain several enzymes which carry out the same reaction. Unfortunately, in many cases these enzymes produce compounds with opposite stereoselectivity. Therefore, whole cells in which such enzymes are present cannot be applied for the synthesis of enantiomerically pure products. To increase the stereoselectivity of a whole cell reaction, recombinant DNA techniques need to be applied. Very common is the overexpression of the enzyme, which catalyzes the particular reaction in a suitable heterologous host such as E. coli. The simultaneous overexpression of an enzyme which catalyzes the regeneration of the consumed cofactor is highly efficient. Ideally, growing cells should provide simultaneously the enzyme for the desired reaction as well as the cofactor regenerating enzyme. Such so-called designer cells seem to be very promising for technical applications. 

Enzyme suppliers determine the activity of their products by measuring the extent of the catalysed reaction under carefully controlled conditions. A standard test exists for amylases (AATCC Test Method 103) , but the evaluation of cellulases is more complex and can vary from supplier to supplier. One common method is to measure the degradation of carboxymethylcellulose solutions. Another is to determine the weight or strength loss of standard cotton fabrics under laboratory conditions where there is a correlation problem, because the mechanical conditions of the technical application are different to the laboratory ones. For example, the hydrolysis degree, HD, is determined by HD = (m - m)/m where and m are the weight of the test material before and after bio-fmishing. 

Surfactant aggregates (microemulsions, micelles, monolayers, vesicles, and liquid crystals) are recently the subject of extensive basic and applied research, because of their inherently interesting chemistry, as well as their diverse technical applications in such fields as petroleum, agriculture, pharmaceuticals, and detergents. Some of the important systems which these aggregates may model are enzyme catalysis, membrane transport, and drug delivery. More practical uses for them are enhanced tertiary oil recovery, emulsion polymerization, and solubilization and detoxification of pesticides and other toxic organic chemicals. 

Decreasing the particle size of the carriers. In technical applications the lower limit is a diameter for spherical particles of > 100 pm, which allows the convenient retention on a sieve plate even in large enzyme reactors. For smaller sized enzyme crystals other retention techniques have to be applied. 

On the other hand, in nature a continuous uptake of substrate and release of product without loss of biocatalysts is not achieved by carrier fixation but by means of cellular membranes. Efficient immobilized enzyme reactor systems for technical applications can therefore be established replacing cellular membranes by ultrafiltration or reverse osmosis synthetic membranes, and the activated transport through the cellular wall by a forced flow across the membrane.7... 

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. 

The term should be used for enzymes that display Michaelis-Menten kinetics. Thus, it is not used with allosteric enzymes. Technically, competitive and noncompetitive inhibition are also terms that are restricted to Michaelis-Menten enzymes, although the concepts are applicable to any enzyme. An inhibitor that binds to an allosteric enzyme at the same site as the substrate is similar to a classical competitive inhibitor. One that binds at a different site is similar to a noncompetitive inhibitor, but the equations and the graphs characteristic of competitive and noncompetitive inhibition don t work the same way with an allosteric enzyme. 

Budtz P (1994) Microbial rennets for cheese making. Novo-Nordisk Technical Report, A-6045a Burgess RR (1969) A new method for the large scale purification of Escherichia coli deoxyribonucleicacid-dependent ribonucleic acid polymerase. J Biol Chem 244(22) 6160-6167 Cao L (2005) Carrier-bound immobilized enzymes. Principles, application and design. Wiley-VCH, Weinheim, 563 pp... 

For industrial uses of enzymes, processes have to be technically and economically feasible. Immobilization of the enzyme, which is usually carried by entrapment within a porous solid matrix, has advantages over soluble enzymes (Pereira et al, 2001 Tramper et al 2001). This includes ease of reuse and enhancement of both enzyme activity and selectivity (D Souza, 2001 Gianfreda and Scarfl, 1991). Immobilized enzymes have applications in various industries and in different reactor configurations. Among these reactors, packed bed reactors have shown promise in many processes. This is widely used with an immobilized enzyme system due to long retention times and ease of operation (Abu-Reesh, 1997). 

The most important technical applications of catalytic hydrolysis and acylation involve technical enzymes, as used in food processing, washing powders, or derace-misations. Especially the latter application has also found significant application in chemical synthesis. The kinetic resolution of chiral, racemic esters, anhydrides, or alcohols relies on the faster conversion of only one substrate enantiomer by the chiral catalyst, whereas the other enantiomer ideally remains unchanged. A special case within kinetic resolutions is the desymmetrization of prochiral mexo-compounds like mera-anhydrides (2) or meso-diols, (5) that requires a selective conversion of one of the two enantiotopic functional groups (carbonyl or OH-group, Scheme 7.1). 

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