Whole multi-enzyme systems

Multi-Enzyme Systems in Whole-Cell Biotransformations and Expression of Redox Systems 55... 

Thermal biosensors have attracted less consideration. Moreover, adverse comments like complicated thermostating, very weak sensitivity or non-specific heating effects have resulted in a poor reputation. Actually, this trend is surprising because thermal biosensors have influenced the whole of biosensor research over and over again (Mosbach, 1991). Especially the enzyme thermistor (ET) has enriched our knowledge about immobilized multi-enzyme systems for signal amplification, the use of immobilized coenzymes and different immobilization techniques. Moreover, ET basic research was decisive for immunosenso-rics or concanavalin-A-based reversible biosensors. Thermal biosensors have multiple advantages ... 

More recently, techniques of direct immobilization of whole microbial cells have been developed either to avoid the need to extract enzymes from microbial cells or to utilize multi-enzyme systems. Since the ecurly 1960 s, we have studied the immobilization of enzymes cuid microbial cells for industrial applications and have succeeded in the industrialization of three asymmetric reactions. In this report, these already industrialized systems and a potential system for which we are planning industrialization will be described. 

Depending on the characteristics of the electron donor, optical techniques and amperometry can be used. The use of the three enzymes, in homogeneous phase, has been applied in combination with pervaporation as sample pretreatment, for the determination of dichlorvos in soil and water samples [382]. Acceptable recoveries for dichlorvos (90-110%) in natural waters and spiked soil samples were reported. The whole procedure offers a sample frequency of 2 h [382]. Microgravimetric measurements of this multi-enzymatic system have also been reported using benzidines (3,3 - diamino-benzidine) as substrate of peroxidase, which is oxidized to an insoluble product [383]. Similarly to the previous example using this technique, the limits of detection achieved for carbaryl and dichlorvos are not as good as those obtained amperometrically. 

The system as a whole is therefore not merely multi-enzymic by happenstance, but necessarily so for both maximum rate of attack on native cellulose and for maximum completeness of conversion to glucose. 

Utilization of whole cells and tissues in biosensor has increasingly been used. Enzyme stability, availability of different enzymes and reaction systems, and characteristics of cell surface are the advantages of using cells and tissues in biosensor designs. Multi-step enzyme reactions in cells also provide mechanisms to amplify the reactions that result in an increase in the detectability of the analytes. The presence of cofactors such as NAD, NADP, and metals in the cells allows the cofactor-dependent reactions to occur in the absence of reagents. (34, 50, 69). However, the diffusion of analytes through cell wall or membrane imposes constraint to this type of biosensors and results in a longer response time compared to the enzyme biosensors. 

Abstract The multi-step enzyme catalysed biosyntheses of monoterpenoid indole and isoquinoline alkaloids are described. Special emphasis is placed on those pathways leading to alkaloids of pharmacological and medicinal significance which have been fully elucidated at the enzyme level. The successful identification and cloning of cDNAs of single enzymes and their application provides great opportunities to develop novel strategies for both in vitro and in vivo alkaloid production in whole plants or tissue cultures, as well as in microbial systems such as Escherichia coli and yeast. 

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