Lipase-catalyzed synthesis of sucrose monoester: Increased productivity by combining enzyme pretreatment and non-aqueous biphasic medium.

Sucrose monocaprate was synthesized by carrying out a lipase-catalyzed transesterification in a non-aqueous biphasic medium. Vinyl caprate was mechanically dispersed into a solution of sucrose in DMSO. The use of DMSO allowed increasing sucrose concentration up to 0.7M (in DMSO). The denaturing effect of DMSO on lipase was avoided by pretreatment of lipase by pH adjustment in the presence of crown ether. This pretreatment maintained a significant catalytic activity which led to 0.2M sucrose monoester within 1h at 50°C, which represented higher productivity than already reported. Detailed structural characterization revealed that only monoester was recovered and the 2-O-acylated sucrose monocaprate was the major isomer in the final product.

Scalable preparation of silCoat-biocatalysts by use of a fluidized-bed reactor.

SilCoat-biocatalysts are immobilized enzyme preparations with an outstanding robustness against leaching and mechanical stress and therefore promising tools for technical synthesis. They consist of a composite material made from a solid enzyme carrier and silicone. In this study, a method has been found to enable provision of these catalysts in large scale. It makes use of easily scalable fluidized-bed technology and, in contrast to the original method, works in almost complete absence of organic solvent. Thus, it is both a fast and safe method. When the Pt-catalyst required for silicone formation is cast on the solid enzyme carrier before coating, resulting composites resemble the original preparations in morphology, catalytic activity, and stability against leaching and mechanical forces. Only the maximum total content of silicone in the composites lies about 10% w/w lower resulting in an overall leaching stability below the theoretical maximum. When the Pt-catalyst is mixed with cooled siloxane solution before coating, surficial coating of the enzyme carriers is achieved, which provides maximum leaching stability at very low silicone consumption. Thus, the technology offers the possibility to produce both composite and for the first time also core-shell silCoat-particles, and optimize leaching stability over mechanical strength according to process requirements.

Toward one-pot lipase-catalyzed synthesis of poly(ε-caprolactone) particles in aqueous dispersion.

The preparation of polyester particles using enzyme-catalyzed (lipase from Candida antarctica B, CALB) ring-opening polymerization of ε-caprolactone (ε-CL) in aqueous dispersion was demonstrated for the first time. Immobilization of CALB enabled a significant increase of the number-average degree of polymerization of ε-CL oligomers (up to 38) as compared to dissolved CALB (8 at the maximum). The nature and amount of lipase, as well as the nature of the support material were identified as key parameters controlling ring-opening polymerization of ε-CL in aqueous dispersion. In addition, the involvement of solubilized monomers in polymerization elementary reactions was demonstrated and the consequences on oligomers average length were detailed. An overall mechanism of lipase-catalyzed ε-CL polymerization in aqueous dispersion taking into account the colloidal nature of reaction medium was proposed on the basis of experimental results.

Encapsulation of synthetically valuable biocatalysts into polyelectrolyte multilayer systems.

Layer-by-Layer (LbL) technology recently turned out to be a versatile tool for the encapsulation of bioactive entities. In this study, the factual potential of this technology to encapsulate synthetically valuable biocatalysts, that is enzymes and whole cells expressing a specific catalytic activity, was investigated. The biocatalysts were embedded into a polyelectrolyte multilayer system involving poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS). The enzymes were adsorbed to CaCO3 or DEAE-cellulose previous to encapsulation. A slight increase (32%) of the catalytic performance was observed for lipase B from Candida antarctica when four layers of polyelectrolytes were applied. On the whole, however, the residual activity of the investigated enzymes after encapsulation was rather low. Similar results were obtained with whole-cell biocatalysts. It was found that the activity decrease can be attributed to mass transfer restrictions as well as direct interactions between polyelectrolytes and catalytically active molecules. Both effects need to be understood in more detail before LbL technology can be advanced to technically efficient biocatalysis.

Colorimetric assay for sensitive poly(styrene sulfonate) quantification in a template directed polyelectrolyte-assembling process.

Template directed Layer-by-layer (LbL) technology recently moved into the center of scientific attention, particularly as a versatile tool for bioencapsulation purposes. Its major advantages can be found in the striking simplicity of tuning wall properties and the complete control over layer thickness and permeability. Yet, for the most commonly applied pair of polyelectrolytes, poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS), the mandatory control of the successful deposition on plane and colloidal surfaces is currently only attainable by means of sophisticated and expensive equipment. Here we describe an alternative quantification method based on a simple colorimetric assay using the Bradford reagent, a cost-effective commercially available dye, and standard laboratory technical devices. The binding of the dye to PSS causes a distinct shift of the absorption maximum from 465 to 680 nm, providing a method for spectral quantification of submicrogram amounts of dissolved PSS during LbL coating with significant accuracy and excellent reproducibility. The method was successfully employed to quantify accurate polyelectrolyte loadings on several particles that have a general importance as LbL templates. Thus, this method can be recommended as standard laboratory technique for control of LbL encapsulation and will considerably broaden the applicability of this promising technology in biotechnology.