Enzymes screening

Subtilisin Carlsberg exhibited virtually absolute specificity ( 200 [35]) for the (S)-ester that corresponds to the L-form of a natural amino acid and provided the desired acid (S)-2 in high enantiomeric and chemical purity. Various commercial enzyme preparations from Bacillus lichmiformis were available the cheaper solid preparations Optimase M 440 from MKC [32] and Alcalase 2.0 T from Novo Nor-disk [33] as well as the more expensive liquid forms Alcalase 2.5 L and Optimase L 660. 

Conditions 2 (1.6 g) was emulsified at 36 °C in 5 mM CaCl2 (25 mL) and the pH adjusted to 7.5. The reaction was started by the addition of the enzyme and the pH kept constant by the controlled addition of 1.0 M NaOH under vigorous stirring. Work-up repeated extraction with ethyl acetate at pH 7.5 and 2.2. 

---

Yazbeck D, Tao J, Martinez C, Kline B, Hu S (2003) Automated enzyme screening methods for the preparation of enantiopure pharmaceutical intermediates. Adv Syn Cat 4 524-532... 

By performing the desymmetrization on a prochiral diol, a far more efficient asymmetric biocatalytic route was subsequently developed. Enzyme screening found that... 

Increase thermostability and environmental compatibility Increase specificity and activity Develop thermophilic enzymes screen for new microbes clone and overexpress in industrial hosts site-direct mutagenesis... 

Enzyme Screening, Optimization, and Recycling of Undesired Enantiomer... 

Among the 50 enzymes screened, Rhizopus arrhlzus mycelial lipase showed the best results in terms of E, which is the enantioselectivity coefficient depending of the enantiomeric excess and the conversion (Fig. 7.) . 

Mediated enzyme screen-printed electrode probes for clinical, environmental and food analysis... 

Mediated enzyme screen-printed electrode probes... 

Different industries pose different challenges to the protein formulator. Many feed enzymes are sold as formulated liquid concentrates. In this case, the major requirements for a liquid formulation are enzymatic stability and preservation against microbial growth. It is sometimes not appreciated that the dominant factor affecting enzyme stability is the intrinsic stability of the enzyme itself formulation can do very little to correct for a structurally labile protein. Therefore, it is advisable to make stability an important criterion of the initial enzyme screening process. 

Fig. 8.2. Stages of enzyme screening. Microorganisms are grown in flasks or deep-well microplates. Separation of single clones is achieved either by FACS or by plating on agar plates and subsequent colony picking into microplates. Finally, assays are carried out in microplate formats using whole bacteria or cell extracts. Alternatively, bacterial clones can be directly screened on the level of colonies on indicator agar plates or during FACS.

The demand for enzyme assays that not only monitor overall activity but also en-antioselectivity stimulated the development of further assay systems that are still, however, in a rather experimental state with respect to high-throughput enzyme screening applications. These methods include assays based on electron spin resonance spectroscopy (ESR) [91], nuclear magnetic resonance (NMR) [92,93], IR-thermography [94] or electrospray ionization spectrometry (ESI-MS) [95]. 

Fig. 8.4. Examples of robotic enzyme screening workstations. A) Linear system (Zymark) B) Circular system (CyBio).

Figure 10.31 Evolution of a natural, RNA-cleaving ribozyme into a DNA-cleaving enzyme screening of the biosynthetic ON ribozyme library L20 and selection of the bios)mthetic ON...

The use of enzymes to stereospecifically form amide bonds has been described in many texts (115) however, the commercial availability cf cross-linked enzyme crystals (CLECs), for example, PeptiCLEC-TR, which is an immobilized form of Thermolysin protease, has been used in the synthesis of D2163 (68), a novel matrix metalloproteinase inhibitor (116). In vitro enzyme screening identified the all-natural SSS-isomer as the active product. The elegant CLEC (117) technology used in this example makes the enzyme stable to typical organic reaction conditions and enables facile removal of the enzyme at the end of the reaction by simple filtration. On this basis, it is... 

---