An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes.

The gene encoding a heat-labile uracil-DNA glycosylase (UDG) from a psychrophilic, gram-positive marine strain (BMTU3346) has been cloned, sequenced, and expressed in Escherichia coli. The UDG is a cold-active enzyme with an apparent temperature optimum of 35 degrees C and a half-life of 2min at 40 degrees C. The amino acid sequence shows an identity of 39.1%-46.2% to UDGs from mesophilic bacteria. The primary structure was examined for features that could be related to the thermolability of the enzyme. The amino acid sequence of the heat-labile UDG shows 22 differences with respect to the consensus sequence derived from bacterial UDGs. Features previously recognized in cold-active enzymes such as extended surface loops or a decrease in the number of arginine residues or proline residues in loops were not observed. Because dominant features that could be related to the thermolability of the UDG from BMTU3346 cannot be identified, more subtle modifications of the conformation seem to be responsible for its thermolability.

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The article contains sections titled: 1. General Production Methods 1.1. Microbial Production 1.1.1. Organism and Enzyme Synthesis 1.1.2. Strain Improvement 1.1.3. Physiological Optimization 1.1.4. The Fermentor and its Limitations 1.1.5. Process Design 1.1.6. Modeling and Optimization 1.1.7. Instrumentation and Control 1.2. Isolation and Purification 1.2.1. Preparation of Biological Starting Materials 1.2.1.1. Cell Disruption by Mechanical Methods 1.2.1.2. Cell Disruption by Nonmechanical Methods 1.2.2. Separation of Solid Matter 1.2.2.1. Filtration 1.2.2.2. Centrifugation 1.2.2.3. Extraction 1.2.2.4. Flocculation and Flotation 1.2.3. Concentration 1.2.3.1. Thermal Methods 1.2.3.2. Precipitation 1.2.3.3. Ultrafiltration 1.2.4. Purification 1.2.4.1. Crystallization 1.2.4.2. Electrophoresis 1.2.4.3. Chromatography 1.2.5. Product Formulation 1.2.6. Waste Disposal 1.3. Immobilization 1.3.1. Definitions 1.3.2. History 1.3.3. Methods 1.3.3.1. Carrier Binding 1.3.3.2. Cross‐linking 1.3.3.3. Entrapment 1.3.4. Characterization 1.3.5. Application 2. Discovery and Development of Enzymes 2.1. Enzyme Screening 2.1.1. Overview 2.1.2. Natural Isolate Screening 2.1.3. Molecular Screening 2.1.4. Environmental Gene Screening 2.1.5. Genomic Screening 2.1.6. Proteomic Screening 2.2. Protein Engineering 2.2.1. Introduction 2.2.2. Application of Protein Engineering in Academia and Industry 2.2.3. Outlook

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