Recombinant biocatalyst

Recently, recombinant biocatalysts obtained using Escherichia coli cells were designed for this process. The overexpression of all enzymes required for the process, namely, hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771 was achieved. These cells were used for production of a-amino acids at the concentration of above 50 g 1 dry cell weight [37]. This is an excellent example presenting the power of biocatalysis with respect to classical catalysis, since a simultaneous use of three different biocatalysts originated from one microorganism can be easily achieved. 

Historically, the most popular enzymes used for chemical synthesis are lipases, esterases, proteases, acylases and amidases, among others. Recently, a number of recombinant biocatalysts have been discovered and isolated, significantly expanding the toolbox for biotransformations. In this section, the focus will be on these new enzymes. 

The bioconversion was carried out in a two-liquid phase system (Figure 15.12), which was developed at the 2-L level, and scaled up to the 30-L level to produce almost 400 g of product. Several apolar phases were used, of which bis(2-ethylhexyl)phthalate (BEHP) was preferred because it showed a better partitioning of epoxystyrene toward the apolar phase and away from the aqueous phase than did hexadecane. This was important because the product was quite toxic to the recombinant biocatalyst when it appeared in the aqueous phase. This bioconversion illustrates that apolar compounds like styrene and its epoxide, which are quite toxic to microorganisms, can be handled successfully in two-liquid-phase cultures. The toxicity of the substrate and product are not significant issues here. 

Design of a Tailormade Recombinant Biocatalyst Including Hydantoin Racemose Enzymes 187... 

The behavior of the recombinant biocatalyst on a larger scale is of considerable economic interest. For this reason D-methionine production was analyzed from 300mM D,L-methylthioethylhydantoin (52.3g/1), in a reaction volume 300 times that used at the laboratory scale (300ml). When system 1 was induced in optimal conditions the product yield of D-methionine reached 100% in 6h and there was no D-carbamoyl-methionine accumulation (Figure 12.15a). However, when the... 

Catalyst optimization, application scope, reaction parameters, case studies Microorganism screening, recombinant biocatalyst, reaction parameters, product isolation... 

The key area of opportunity is improved and novel biocatalysts. The search is on to examine the remaining unexplored biological diversity which presumably holds a great wealth of biocatalytic potential and of new bioactive compounds and biomaterials. The introduction of biotechnology into many industrial processes will be increasingly dependent on the development of recombinant biocatalysts. 

Previous works have examined the application of S. cerevisiae as a recombinant biocatalyst [102], Geerlings et al. [102] reported the construction of a S. cerevisiae strain harboring two cDNAs coding for Strictosidine synthase and Strictosidine P-glncosidase from the medicinal plant C. roseus. 

A. Schmid, H.-P.E. Kohler, K.-H. Engesser, E. coli 1M109 pHBP461, a recombinant biocatalyst for the regioselective monohydroxylation of ortho-substituted phenols to their corresponding 3-substituted catechols, 1. Mol. Catal. B Enzym. 5 (1-4) (1998) 311-316. 

In particular, the availability of such bacterial biocatalysts in the form of recombinant expression systems [136] in combination with simplified purification protocols opened up this methodology for large-scale applications [204]. 

The biocatalytic approach is based on recombinant Escherichia coli growing in an aqueous mineral medium (Scheme 5.4). In Scheme 5.4, microbial growth is translated into a stoichiometric equation for biocatalyst synthesis. One needs to consider that biological safety regulations for recombinant class 1 organisms (no danger for humans and the environment) have to be followed with respect to biocatalyst handling. 

Park, J.B., Buehler, B., Habicher, T. etal. (2006) The Efficiency of Recombinant Escherichia coli as Biocatalyst for Stereospecffic Epoxidation. Biotechnology and Bioengineering, 95(3), 501-512. 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

Recombinant Whole-Cell Biocatalysts Overexpressing Catalytic Enzymes... 

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

In summary, ketoreductases have emerged as valuable catalysts for asymmetric ketone reductions and are preparing to enter the mainstream of synthetic chemistry of chiral alcohols. These biocatalysts are used in three forms wild-type whole-cell microorganism, recombinant... 

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