Biocatalyst techniques

Prerequisites for the large-scale applications are a rapid production technology for the biocatalysts, techniques for the stabilization of the biocatalyst and the use of the stable biocatalysts in appropriate reactors. 

Here we will focus on the biochemical aspects. The techniques of isolating enzymes, the process of enzyme immobilisation and the behaviour of immobilised enzyme reactors are discussed in detail in the BIOTOL text Technological Applications of Biocatalysts", so will not deal with these aspects in detail here. In outline, however, once the desired enzyme is isolated, it is attached to a carrier material. In order to ascertain sufficient accessibility of the enzyme, a bifunctional spacer molecule is attached to the carrier ... 

Efficiency and selectivity are the two keywords that better outline the outstanding performances of enzymes. However, in some cases unsatisfactory stereoselectivity of enzymes can be found and, in these cases, the enantiomeric excesses of products are too low for synthetic purposes. In order to overcome this limitation, a number of techniques have been proposed to enhance the selectivity of a given biocatalyst. The net effect pursued by all these protocols is the increase of the difference in activation energy (AAG ) of the two competing diastereomeric enzyme-substrate transition state complexes (Figure 1.1). 

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... 

Use of molecular biology technique to find novel biocatalyst... 

Abstract Alkaloids are very much important molecules, not only for chemical reasons but also because of their diverse biological activities. Up to now several reviews have been published explaining the use of biotransformation or microbial transformation techniques to modify alkaloids, which added several advantages over the classical chemical transformation systems. This chapter is a critical update of the microbial transformations reported in the last couple of years, targeting novel biocatalysts from microbes. 

Despite advent of theoretical methods and techniques and faster computers, no single theoretical method seems to be capable of reliable computational studies of reactivities of biocatalysts. Ab initio quantum mechanical (QM) methods may be accurate but are still too expensive to apply to large systems like biocatalysts. Semi-empirical quantum methods are not as accurate but are faster, but may not be fast enough for long time simulation of large molecular systems. Molecular mechanics (MM) force field methods are not usually capable of dealing with bond-breaking and formation... 

In this section, we will consider the methodologies used for genetic engineering of biocatalysts for desulfurization and the biocatalysts developed so far via various technologies. The application of genomic techniques as reported in patent literature associated with BDS is described first. 

Despite all the intellectual property generated in this field, the application has not reached commercial scale. It does not mean that there has not been any progress. In fact, the biocatalyst development has greatly advanced, much of it due to the advancements in the techniques, methods and tools related to MB and GE. MB techniques raised the understanding of the biocatalyst from the level of whole cells to clearly defined... 

The methods developed by EBC and others in the late 1990s using hydrocyclones and phase-inversion techniques may be sufficient for separation of the treated oil from the aqueous phase and biocatalyst. However, a cost analysis of such methods may be necessary to determine the economic feasibility. Recent work using hydrophobic membranes, magnetically separable immobilized biocatalysts and other techniques may provide alternate methods for separation of oil and recycling biocatalyst. A comparison of these techniques with each other and the previously investigated hydrocyclone techniques is needed to demonstrate improvements in the separation efficiency. 

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