No abstract

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.

The mechanical and leaching stability of enzymes adsorbed on macroporous carriers is an important issue for the technical applicability of such biocatalysts. Both can considerably benefit from the deposition of silicone coating on the carrier surface. The coating of the immobilized lipase Novozyme 435 (NZ435), as a model enzyme preparation, with different silicone loadings was studied in detail by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as well as by energy‐dispersive X‐ray spectroscopy (EDX) and BET isotherms, and offers explanations and prerequisites for its stabilizing effects. The deposition of silicone on the poly(methyl methacrylate) (PMMA) carrier was found to form an interpenetrating network composite rather than the anticipated core‐shell structure. The silicone precursors homogeneously wet the carrier surface including all inner pores and gradually fill the complete carrier. In parallel, the surface area of NZ435 decreases from an initial value of 89 m2g−1to 0.2 m2g−1after silicone loading. A visible layer of silicone on the outer surface of the carrier was only observed at a silicone concentration of 54 % w/w and more. Maximum leaching stability corresponds to the formation of this layer. The mechanical stability increases with the amount of deposited silicone. It can be expected that stabilization against leaching and/or mechanical stress by formation of silicone composites can easily be transferred to a whole range of alternative biocatalytic systems. This should considerably advance their general technical applicability and overall implementation of biocatalysts in chemical synthesis.

Due to favourable partition coefficients the highly enantioselective reduction of 2-octanone, catalysed by an alcohol dehydrogenase from Lactobacillus brevis, is faster in a biphasic system containing buffer and the ionic liquid [BMIM][(CF(3)SO(2))(2)N] compared to the reduction in a biphasic system containing buffer and methyl tert-butyl ether.

What is the best solvent combination for a catalytic biphasic reaction system to maximise equilibrium conversion? And which phase volume ratio and reaction stoichiometry maximises the conversion? Which product yield can be obtained using a distinct biphasic reaction mixture? These questions are essential, as optimisation can only be carried out within the thermodynamically imposed boundaries. Within this paper we introduce a mathematical expression to calculate and predict equilibrium conversion and product yield in biphasic reaction mixtures with only one reactive phase. This mathematical expression allows a time-saving design of biphasic experiments with respect to maximum conversion and product yield. The calculations were verified by experimental data obtained from a substrate-coupled alcohol dehydrogenase-catalysed reduction under different biphasic reaction conditions.