Genetically-modified bacteria and uses thereof

A genetically-modified bacterium, for example of the class Actinobacteria, and the use of such a bacterium in the bioconversion of a steroidal substrate into a steroidal product of interest. A method of converting a steroidal substrate into a steroidal product of interest, wherein the method comprises: inoculating culture medium with genetically-modified bacteria according to any of Claims 1 to 28 and growing the bacterial culture until a target OD600 is reached; adding a steroidal substrate to the bacterial culture when the target OD600 is reached; culturing the bacterial culture so that the steroidal substrate is converted to the steroidal product of interest; and extracting and/or purifying the steroidal product of interest from the bacterial culture

Exploiting enzyme promiscuity for rational design

Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area. Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity. In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant. In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.QC 2010092

Chitosan flocculation: An effective method for immobilization of E. coli for biocatalytic processes.

Immobilization of Escherichia coli cells containing a ω-transaminase was carried out by flocculation with chitosan and the preparation was used in asymmetric synthesis of (S)-4'-cyano-α-methylbenzylamine, recycled in five consecutive batches. Chitosans with different molecular weights and degrees of acetylation were compared and effects of varying the chitosan properties, cell concentration and ratio of cells to chitosan were studied. Immobilization was achieved by increasing the pH to slightly alkaline, which induced the formation of large fast sedimenting flocs. Although an effective immobilization was obtained using most types of chitosan, high molecular weight and low degree of acetylation were considered favourable properties, resulting in good floc stability and quick sedimentation. It was found that it was possible to affect the floc characteristics, by changing the ratio of cells to chitosan in such a way that preparations resembling either entrapped or cross-linked cells could be obtained. The volume of the sedimented preparation decreased approximately 50% when increasing the cell to chitosan ratio from 2g/g to 10g/g at a constant amount of cells. Despite very high concentrations of cells (10-100g cells/g chitosan) in the flocculated preparations, diffusion limitations were minimal. Flocculation with chitosan was considered a simple and effective method for immobilization of E. coli cells for biocatalytic processes

Activity and stability of different immobilized preparations of recombinant E. coli cells containing omega-transaminase

Production of chiral amines using omega-transaminases has been thoroughly studied in recent years. Immobilized w-transaminases, however, have been used on relatively few occasions despite potential benefits such as reuse of enzyme and ease of product purification. In this study principally different methods including surface immobilization, entrapment and sweep flocculation using titanium oxide. Ca-alginate and chitosan respectively were evaluated for the immobilization of recombinant Escherichia coil cells. The enzyme expressed was a modified Arthrobacter citreus omega-transaminase with improved thermostability. The preparations were compared in terms of cell loading capacity, operational stability in repeated batches and storage stability using the conversion of methylbenzylamine to acetophenone. The use of chitosan for cell immobilization proved to be the method of choice since it was both very simple and effective. At a very high cell loading of 3.2 g cells/g chitosan >60% activity was observed. The preparation was reused in eight successive 1-h batches with >90% remaining activity. To further demonstrate its usability the preparation was used for asymmetric synthesis of (S)-4'-cyano-(alpha)-methylbenzylamine in three repeated bathes (cycle time >20 h), using isopropylamine as the amine donor. Storage stability was comparable with that of non-immobilized cells. It was concluded that the chitosan method due to its properties and simplicity would be advantageous for use also on a larger scale. (c) 2012 Elsevier Ltd. All rights reserved