The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure

At low water activity alpha-chymotrypsin is more active in an ionic liquid than in non-ionic organic solvents

The kinetics of the alpha-chymotrypsin catalysed transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-butanol and the competing hydrolysis were evaluated at fixed water activity in two ionic liquids and two non-ionic organic solvents. In most respects the four solvents behaved similarly. However, at a water activity of 0.33, higher catalytic activity was observed in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide, than in the other solvents, and at a(w)=0.11 catalysis was only observed in this solvent

St. John's Daily Star, 1919-03-04

The St. John's Daily Star was published daily except Sunday between 17 April 1915 - 23 July 1921

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

International audienceSilCoat-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

In situ microscopy for in-line monitoring of the enzymatic hydrolysis of cellulose

A new in-line method for the monitoring of enzymatic hydrolysis of cellulose is described. Using a new in situ microscope prototype, the noninvasive determination of particle size distributions was possible. For the automated analysis of the acquired images, a new processing algorithm called CelluloseAnalyzer was developed. It enabled tracking of the number of particles and moreover allowed monitoring of the proportions of particle size fractions during the course of enzymatic hydrolysis reactions. Using this technique, significant differences between hydrolysis with endoglucanases and cellulase mixtures were observed. Furthermore, the in situ microscopy results were compared with results from off-line measurements with laser diffraction spectroscopy and gel permeation chromatography. © 2013 American Chemical Society