Biocatalyst development

Initially, conventional selection methods were used to select aspartase overproducing strains. For example. Nishimura and Kisumi isolated aspartase hyperpro- 

The cloning of the aspA gene onto the multicopy plasmid pBR322 [29] led to a modest fourfold to sixfold overproduction of aspartase compared with host strain without the clone [24]. The aspartase activity of the clone was further enhanced by deregulation of the gene using the lactose inducible, tac promoter [30,31]. The resultant plasmid (pPT174), when fully induced, produced 190-fold more aspartase activity than the E. coli K12 parental strain without the plasmid [31], 

The Mitsubishi Chemical Company has described a process for the commercial production of L-aspartate using an cx-amino-zr-butyric acid resistant mutant of B. flavum [11]. The enzyme is moderately thermal resistant, allowing the process to be run at 45°C. The process is run using immobilized cells in a fed batch system in which the biocatalyst is recycled [4]. An initial problem was the conversion of fumarate to malic acid by an intracellular fumarase activity, which led to low l-aspartic acid yields during the first cycle. This problem was circumvented by preheating the biocatalyst for 1 hour at 45°C, which completely destroyed the fumarase activity [4,11]. Recently, the aspartase gene from B. flavum has been cloned [28] and has presumably been used to improve the efficiency of this process. 

Plasmid Total activity Protein expression Specific activity I 

Total activity normalized to wild type aspartase expressed using the tac promoter. 

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Prepolymer method for entrapment of biocatalyst developed by Fukui and Tanaka is considered to be one of the best immobilization methods. Characteristic features and advantages of the prepolymer method can be summarized as follows ... 

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... 

In an in depth comparison of the cumulative knowledge discussed in Chapter 3, with what one could extract from the technological results reported in this Chapter, perhaps the first observation that one can make is the difference between the content of the biocatalyst development vs. process development results. The results on biocatalyst improvements constitute the majority of the open literature reports. The most important bottleneck holding advancement of the biodesulfurization technology is the ability to break the second C-S bond, releasing the sulfur from the organosulfur molecules. The IP portfolio does not provide a real solution for that problem. 

Developing a bioprocess for removing heteroatoms from petroleum or for upgrading heavy crudes involves two main components. The first is biocatalyst development and second is bioprocess development. Both of these topics are discussed in this book,... 

In other words, biocatalysis should be not considered by itself but integrated in the whole bio-process (Figure 2.16a). Substantially increased emphasis on biocatalyst development is an important goal for chemistry-related industries, even if biocatalysts carmot reach their potential without a concerted eflfort on the parallel development of other components of bioprocessing, as well as an integration with... 

Concerning the biocatalyst development by directed evolution, some limitations of naturally occurring enzymes such as thermostability, activity, and tolerance towards organic solvents have been successfully overcome. It is also important to mention that this methodology is highly effective in altering, or improving almost all kinds of protein properties for different classes of enzymes. 

Wang M, Si T, Zhao H (2012) Biocatalyst development by directed evolution. Bioresour Technol 115 117-125... 

Life cycle inventory data for the production of PDO are generated from a detailed process model for bioproduction from glucose. The process design is developed from publicly available data on the biocatalyst developed by DuPont... 

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