Competition of Thermomyces lanuginosus lipase with its hydrolysis products at the oil-water interface.

Lipase-catalyzed hydrolysis of triglycerides yields glycerol and free fatty-acids, provided that the enzyme is non-regioselective. For an Sn-1,3 regioselective enzyme, such as lipase from Thermomyces lanuginosus, the final product is no longer glycerol but Sn-2 monoglyceride instead. However, surface active molecules generated by lipolysis may have a detrimental effect on the interfacial biocatalysis since it is known that low molecular weight surfactants can displace proteins from interfaces. By using drop profile analysis tensiometry, we evaluated the interfacial properties of the lipase-generated molecules and their competitive effect on the adsorption behavior of the lipase and on the proceeding lipolysis. Our results show that even at concentration ratios of 8.64×10-4M (Sn-2 monoglyceride) to 2.5×10-7M (lipase), the final interfacial pressure values are very similar as for the system containing the lipase alone (i.e. ∼26 mN/m). This is a strong indication that monoglycerides, as the most interfacially active products generated during regioselective lipolysis, are expelled from the oil-water interface by the lipase. We attribute this effect to intermolecular lipase-lipase interactions, resulting in a low desorption probability of the lipase. For low oleic acid concentrations, the interfacial tension is solely determined by the lipase, while for higher concentrations, lipase and oleic acid both contribute to the tension values. We propose a hypothesis based on the preferential interaction of oleic acid molecules with hydrophobic sites on the lipase. The pH dependence of the adsorption rate and the interfacial activity of the lipase were also investigated.

Ellipsometric study of molecular orientations of Thermomyces lanuginosus lipase at the air-water interface by simultaneous determination of refractive index and thickness.

Ellipsometric studies of very thin organic films suffer from the low refractive index contrast between layer and bulk substrate. We demonstrate that null ellipsometry can not only provide detailed information about the adsorption kinetics and surface excess values, but in addition on layer thicknesses with submonolayer resolution of a lipase from Thermomyces lanuginosus at the air-water interface. While measuring very close to the Brewster angle, refractive indices and layer-thicknesses can both be determined with a precision that is sufficiently high to make conclusions on the density and orientation of the molecules at the interface. The orientation was found to be concentration- and pH value-dependent. At the isoelectric point, the lipase was almost vertically oriented with respect to the surface, while for pure distilled water and low lipase concentration a rather horizontal alignment was found. Further experiments, varying the size of the interfacial area in a Langmuir trough, confirm the different layer structures.

Impact of mass transport on the enzymatic hydrolysis of rapeseed oil.

In order to assess the capillary segmented flow reactor as a potentially appropriate reactor device for the enzymatic hydrolysis of vegetable oils, a study was made to reveal the impact of incident mass transfer processes on the hydrolysis rate. As demonstrated by means of experiments performed in a modified Lewis-cell type contactor, which allows the independent adjustment of flow rates for both phases, the enzymatic hydrolysis rate of rapeseed oil is strongly governed by mass transport processes taking place in both phases. In the oil phase, any increase in convective mass transfer results in an enhancement of hydrolysis rate due to facilitated removal of fatty acids from interface layer which is known to inhibit the activity of the enzyme adsorbed at the interface. At asynchronous condition when solely the water phase is agitated, however, convective mass transport in the interface layer has an inverse effect on the hydrolysis rate due to the generation of considerable shear stress in the vicinity of the interface unfavorable for the performance of the enzymes. By operating at synchronous agitation conditions, the shear stress can considerably be reduced. Generally, the positive effect of mass transport in the oil phase compensates the negative one in the aqueous phase thus resulting in an overall increase in hydrolysis rate of 57% with increasing stirrer rates. The results can be applied to the operation of segmented-flow capillary reactors by choosing the oil phase as disperse phase and the water phase as continuous phase, respectively.

Combining the effects of pulping severity and alkali concentration to optimize the lignocellulose-based AlkaPolP biorefinery concept.

Within the framework of the development of a novel lignocellulose biorefinery concept alkaline polyol pulping (AlkaPolP) of Pinus sylvestris was performed at different alkali concentrations. The obtained experimental data were used to develop simple mathematical models that allow the prediction of product yields and properties in dependence on a single parameter combining the effects of time, temperature and catalyst concentration. For this purpose the usual approach expressing the pulping severity R0 had to be complemented by the alkali concentration resulting in a modified severity factor R0('). The found regression models in the form of functions f(R0(')) can be used as a tool for the identification of those pulping conditions giving the desired product characteristics. Because the yields of the biorefinery products reach their maxima at different pulping conditions the optimization of the whole process turned out to be a multi-objective optimization problem.

Dry entrapment of enzymes by epoxy or polyester resins hardened on different solid supports.

Embedding of enzymes was performed with epoxy or polyester resin by mixing in a dried enzyme preparation before polymerization was started. This fast and low-cost immobilization method produced enzymatically active layers on different solid supports. As model enzymes the well-characterized Thermomyces lanuginosus lipase and a new threonine aldolase from Ashbya gossypii were used. It was shown that T. lanuginosus lipase recombinantly expressed in Aspergillus oryzae is a monomeric enzyme with a molecular mass of 34kDa, while A. gossypii threonine aldolase expressed in Escherichia coli is a pyridoxal-5'-phosphate binding homotetramer with a mass of 180kDa. The enzymes were used freeze dried, in four different preparations: freely diffusing, adsorbed on octyl sepharose, as well as cross-linked enzyme aggregates or as suspensions in organic solvent. They were mixed with standard two-component resins and prepared as layers on solid supports made of different materials e.g. metal, glass, polyester. Polymerization led to encapsulated enzyme preparations showing activities comparable to literature values.

Alkaline polyol pulping and enzymatic hydrolysis of hardwood: effect of pulping severity and pulp composition on cellulase activity and overall sugar yield.

The saccharification of beech wood using alkaline polyol pulping (AlkaPolP) and enzymatic hydrolysis was investigated. It will be demonstrated that the AlkaPolP process yields high quality pulps which can easily be hydrolyzed by cellulases. In order to find optimum reaction conditions chips of Fagus sylvatica were pretreated by alkaline glycerol at temperatures between 190 and 230 °C for 15, 20, and 25 min. The impacts of temperature and time were expressed using a severity factor R0. The dependencies of the conversion during enzymatic hydrolysis on severity, pulp yield, delignification and pulp composition are shown. In further experiments it was investigated if the sugar yields can be increased by the application of ultrasound or surfactants before enzyme addition. Up to 95% of the initial cellulose in wood were converted into glucose using cellulases from Trichoderma reesei and ß-glucosidase from Aspergillus niger.

Alkaline polyol pulping and enzymatic hydrolysis of softwood: effect of pulping severity and pulp properties on cellulase activity and overall sugar yield.

The saccharification of softwood using alkaline polyol pulping (AlkaPolP) and enzymatic hydrolysis was investigated. It will be demonstrated that the AlkaPolP process yields high quality pulps which can easily be hydrolyzed by cellulases. Temperature (180-230°C) and duration (15-60 min) of the alkaline glycerol pulping, expressed as pulping severity R0, were varied to find optimum reaction conditions. The obtained pulps were characterized regarding their residual lignin content, kappa number and crystallinity index. Thus, the dependencies of the conversion during enzymatic hydrolysis on severity, pulp composition and pulp characteristics could be observed. In further experiments it was investigated how the enzymatic hydrolysis is affected by pulp drying or by a reduction of enzyme loading. Up to 83% of the initial cellulose in wood and almost 97% of the cellulose in pulp were converted into glucose using cellulases from Trichoderma reesei and ß-glucosidase from Aspergillus niger.