Whole-cell biocatalysts applications

The commercial availability of enzymes or whole cell biocatalysts for a desired biotransformation is freqnently a limiting factor for commercial application of biocatalysts. Enzymes that are cheaply available are typically used in detergents, processing of food, feed and textiles, as well as in waste management applications. Most of these are hydrolytic enzymes, bnt also isomerases (e.g. glucose isomerase) and oxidorednctases are used on indnstrial scale (Table 5.1). 

Hydantoinase-Carbamoylase System for t-Amino Acid Synthesis Despite a number of reports of strains with L-selechve hydantoin-hydrolyzing enzymes [38] the commercial application of the hydantoinase process is stiU restricted to the production of D-amino acids. Processes for the production of L-amino acids are Umited by low space-time yields and high biocatalyst costs. Recently, a new generation of an L-hydantoinase process was developed based on a tailor-made recombinant whole cell biocatalyst. Further reduction of biocatalyst cost by use of recombinant Escherichia coli cells overexpressing hydantoinase, carbamoylase, and hydantoin racemase from Arthrohacter sp. DSM 9771 were achieved. To improve the hydan-toin-converting pathway, the level of expression of the different genes was balanced on the basis of their specific activities. The system has been appUed to the preparation of L-methionine the space-time yield is however still Umited [39]. Improvements in the deracemization process from rac-5-substituted hydantoins to L-amino acids still requires a more selective L-hydantoinase. 

Very recently, however, a remarkably improved whole-cell biocatalyst coexpressing an L-carbamoylase, a hydantoin racemase, and an L-hydantoinase has been developed which showed a 50-fold higher productivity and is suitable for large-scale applications [17]. A prerequisite of this efficient whole-cell biocatalyst, which is applied at Degussa, was the successful inversion of the enantiospecificity of a previously D-selective hydantoinase by means of directed evolution [18]. These improvements have already been confirmed at a m3-scale using a batch reactor concept [17]. 

Fukuda and coworkers have described one of the first applications of the enantioselective hydrolysis of nitriles (Scheme 12.1-6). Using a whole cell biocatalyst optically pure a-hydroxy acids (L-a-hydroxyisovaleric acid and L-a-hydroxyisocaproic acid) have been prepared from the racemates of the corresponding a-hydroxyni-... 

The aminopeptidase from Pseudomonas putida ATCC 12633 has also recently been cloned and overexpressed in E. coli resulting in a highly efficient whole-cell biocatalyst for industrial applications 1291. The specific activity of this new biocatalyst is substantially increased (25 times) compared with the specific activity of the P. putida wild type cells without changing the other positive characteristics of the aminopeptidase. Even though the aminopeptidase from Pseudomonas putida exhibits the relaxed substrate specificity described above, an a-hydrogen atom in the substrate is an essential structural feature for the enzymatic activity. Therefore this enzyme can not be used for the resolution of higher substituted amino acids. 

Whole-cell, hollow-fiber MBR are still under development. Despite their significant potential they have, so far, found only limited application for biochemicals production. One of the reasons is that cleaning of the hollow-fiber membranes is difficult, especially when whole-cell biocatalysts are immobilized in the small fibers. The mass transfer between the nutrients and cells has also to be taken into consideration and enhanced. Immobilizing the biocatalysts in porous beads, instead of directly on the membrane, may tend to avoid some of these problems, and to simplify membrane cleaning. The concept of using MBR as bioartificial organs is technically very attractive the various MBR under development, however, must still be validated with clinical results. One can expect, however, that their development will follow the success of artificial kidneys, which are currently employed worldwide. 

Martinez I, Markovits A, Chamy R et al. (2004) Lipase-catalyzed solvent-free transesterification of wood sterols. Appl Biochem Biotechnol 112 55-62 Matsumoto T, Takahashi S, Kaieda M et al. (2001) Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production. Appl Microbiol Biotechnol 57(4) 515-520 Maurer K (2004) Detergent proteases. Curr Opin Biotechnol 15 330-334... 

Enzyme technology is the application of free enzymes and whole-cell biocatalysts in the production of foods and services. A more narrow definition limits enzyme technology to the technological concepts that allow the use of enzymes in competitive large-scale bioprocesses. Enzyme technology is an interdisciplinary field, recognized by the Organization for Economic Cooperation and Development (OECD) as an important component of sustainable industrial development. 

The invention of new methods of protein engineering in the past decade has led to the construction of an abundance of P450 mutants with new tailored properties. Recent major achievements include significant increases in productivities, yields, and rates of catalytic turnover as well as modification of substrate specificity and efficient multistep reactions in whole-cell biocatalysts, coming one step closer to the technical application of cytochrome P450 monooxygenases. 

Application cf Whole-Cell Biocatalysts Possessing Mutant PARs and LSADH 1165... 

Matsuyama, A., Yamamoto, H., and Kobayashi, Y. (2002) Practical application of recombinant whole-cell biocatalysts for the manufacturing of pharamaceutical such as chiral alcohols. Org. Process Res. Dev., 6, 558-561. 

Enzymes with different stereochemical preferences for 3-oxo esters and 2-alkyl 3-0X0 esters have been isolated [57,62,63,68]. They are NADPH-dependent enzymes and are able to catalyze the reduction of oxo esters of different type. However, they are not available for enzyme-catalyzed reaction in substitution of the whole-cell catalyst. Synthetic applications make use of whole-cell biocatalysts. Valuable intermediates in synthesis are keto esters possessing additional functionality. Thus 5 -4-chloro-3-hydroxybutanoic acid 33 (Scheme 12) has been obtained by reduction with suspended cells from cultures of G. candidum. The compound is the intermediate in the synthesis of the cholesterol antagonist 34. In the biotransformation process a reaction yield of 95% and optical purity of 96% were obtained at 10 g/L. The optical purity was increased to 99% by heat treatment of cell suspensions prior to conducting the bioreduction [69]. 

Chiral epoxides and their corresponding vicinal diols are very important intermediates in asymmetric synthesis [163]. Chiral nonracemic epoxides can be obtained through asymmetric epoxidation using either chemical catalysts [164] or enzymes [165-167]. Biocatalytic epoxidations require sophisticated techniques and have thus far found limited application. An alternative approach is the asymmetric hydrolysis of racemic or meso-epoxides using transition-metal catalysts [168] or biocatalysts [169-174]. Epoxide hydrolases (EHs) (EC 3.3.2.3) catalyze the conversion of epoxides to their corresponding vicinal diols. EHs are cofactor-independent enzymes that are almost ubiquitous in nature. They are usually employed as whole cells or crude... 

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