Screening,Molecular Cloning,and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β‐Hydroxy‐β‐trifluoromethyl Ketone

The stereoselective synthesis of chiral 1,3‐diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β‐hydroxy‐β‐trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2‐trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β‐hydroxy‐β‐trifluoromethyl ketone was identified after purification and subsequent MALDI‐TOF mass spectrometric analysis. As a result, a new NADP⁺‐dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β‐hydroxy‐β‐trifluoromethyl ketone to its corresponding 1,3‐diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N‐ or C‐terminal His‐tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N‐terminal His‐tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β‐hydroxy‐β‐trifluoromethyl ketone.     

Diazoverbindungen. 72. (Diazoalkyl)phosphane – Synthese durch elektrophile Diazoalkansubstitution und oxidative Additionsreaktionen am Phosphor

Diazo Compounds. 72. Diazoalkylphosphanes – Synthesis by Electrophilic Diazoalkane Substitution and Oxidative Addition Reactions at Phosphorus Electrophilic diazoalkane substitution of the diazomethyl compounds 1a,b with the chloro phosphanes 2a-o in the presence of lithium diethylamide yields the diazoalkyl phosphanes 3a-z . Oxidative addition of oxygen, sulfur and selenium at phosphorus leads into the series of oxo, thioxo and selenoxo phosphanes having diazoalkyl substituents ( 4a-d, 5a-m and 7a-d ). The silyl group of 5n,o is cleaved by chromatography on aluminium oxide to yield the (diazomethyl)phosphane sulfides 6a,b .     

Starch-Derived Products for Detergents

Recent developments in the formulation of detergents have been driven by a strong consumer demand for natural and biodegradable products. Detergent manufacturers responded to this demand with corresponding products and advertising slogans such as “fully biodegradable”, “natural” or even “double natural” to oust their competitors. In a detergent formulation, starch- derived products can in principle be used for the following purposes: as the hydrophilic head group in surfactants, as the starting material for (poly)carboxylate co-builders and as the backbone of bleaching activators. Non-ionic classical surfactants can be replaced by alkylpolyglucosides (APGs), a class of products completely based on renewable resources such as glucose and fatty alcohols derived from natural fatty acids. Sodium tripolyphosphate (STPP), the product responsible for the eutrophication of surface waters, can be substituted by a combination of an inorganic zeolite and highly oxidised starch (dicarboxylic starch) or by citrate. Acetylated polyols derived from hydrogenated carbohydrates such as sorbitol can take over the function of the petrochemically-based tetraacetyl ethylene diamine (TAED) used as activator to allow perborate bleaching at lower washing temperatures.     

Erythritol,a New Raw Material for Food and Non-food Applications

In response to an increasing demand from consumers for healthier and calorie controlled foods, Cerestar has developed a new food ingredient, erythritol. Erythritol can be produced from starch by a full biotechnological process, combining enzymatic and fermentative conversions. The use of an osmophilic yeast allows the fermentation step to be performed at high dry substance, giving an economic advantage. An extremely pure end product is then easily obtained by final crystallisation. Erythritol is structurally a polyol and shares the health properties of other polyols such as being toothfriendly and safe for diabetics. However, it offers in addition two very important nutritional advantages: a lower calorific value (0.3 Kcal/g) and a good tolerance. This is due to its low molecular weight, which allows erythritol to be rapidly absorbed from the small intestine, with subsequent excretion in the urine. Fermentation in the colon is therefore excluded and any resulting gastro-intestinal discomfort avoided. The combination of these properties makes erythritol a unique low calorie bulk sweetener. From a functionality point of view, erythritol is a moderately sweet bulking agent with a cooling taste: workability is similar to other polyols. It has a taste profile close to that of sucrose and may therefore improve the taste quality of a blend with intense sweeteners. Its low solubility and ease of crystallisation, make erythritol very suitable for applications which require a crystalline sweetener, such as chocolate. Other potential application areas are bakery, table-top and confectionery. Presently erythritol is under evaluation to establish beyond any doubt its safety and to obtain food approval as a new, low calorie, bulk sweetener. Potential non-food applications of erythritol are in polymers, fine chemicals and pharmaceutical intermediates.     

The Effect of Antibacterial Particle Incorporation on the Mechanical Properties,Biodegradability, and Biocompatibility of PLA and PHBV Composites

The composites based on polylactide (PLA) and poly (3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) with the addition of antibacterial particles: silver (Ag) and copper oxide (CuO) are characterized. Basic mechanical properties and biodegradation processes, as well as biocompatibility of materials with human cells are determined. The addition of Ag or CuO to the polymers do not significantly affect their mechanical properties, flammability, or biodegradation rate. However, several differences between the base materials are observed. PLA‐based composites have higher tensile and impact strength values, while PHBV‐based ones have a higher modulus of elasticity, as well as better mechanical properties at elevated temperatures. Concerning biocompatibility, each of the tested materials support the growth of fibroblasts over time, although large differences are observed in the initial cell attachment. The analysis of hydrolytic degradation effects on the structure of materials shows that PHBV degrades much faster than PLA. The results of this study confirm the good potential of the investigated biodegradable polymer composites with antibacterial particles for future biomedical applications.     

Structure‐Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica

Hydratases provide access to secondary and tertiary alcohols by regio‐ and/or stereospecifically adding water to carbon‐carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure‐based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two‐electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio‐ and stereoselective hydration.