The hydrophobins HFBI and HFBII from Trichoderma reesei showing efficient interactions with nonionic surfactants in aqueous two-phase systems.

Fungal hydrophobins are a group of surface active, self-assembling proteins. The filamentous fungus Trichoderma reesei produces two (class II) hydrophobins, HFBI and HFBII. We have studied how these water-soluble hydrophobins behave in two-phase systems using a series of nonionic surfactants with different characteristics. It was found that both hydrophobins, but especially HFBI, had a very high affinity for the surfactants. The highest partitioning coefficient, over 2500, was observed for HFBI with C(11)EO(2). Reducing the disulfides in the protein resulted in a complete loss of affinity for the surfactant, which demonstrates that the interaction is dependent on the disulfide-stabilized conformation. The hydrophobins could be efficiently extracted back from the surfactant phase by addition of alcohols such as isobutanol. Effects of the type of surfactant, temperature, pH, and ionic strength were investigated. The use of this method for purifying the proteins from crude fungal culture supernatants is demonstrated and implications of the protein-polymer interaction are discussed.

Parameters influencing protein extraction for whole broths in detergent based aqueous two-phase systems.

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed.

Physical properties of detergent-based aqueous two-phase systems.

The physical behavior of the binary phase systems of the non-ionic polyoxyethylene detergent Agrimul NRE 1205 and water was investigated. This technical detergent can be used for the large-scale recovery of biomolecules in detergent based aqueous two-phase systems. The phase diagram was determined. It shows significant and unexpected differences to highly purified detergents. Very similar to neat detergents the phase diagram can be influenced by auxiliary chemicals thus shifting the entire phase diagram in general to lower temperatures. This was demonstrated by lowering the cloud-point by various additions. The concentration factor, as an important parameter of a first capture step in purification was investigated and modeled. Auxiliary chemicals, temperature change and change in detergent concentration also influence the viscosity and density of the phases. These experimental data are shown. They can help to explain the separation behavior of proteins. In large-scale separations aqueous two-phase systems are separated using disc-stack centrifuges. It is demonstrated that this is not a feasible method for detergent-based aqueous two-phase extraction and the physical reason is presented.

Investigation of mathematical methods for efficient optimisation of aqueous two-phase extraction.

Mathematical strategies were applied to optimise the extraction of recombinant leucine dehydrogenase from E. coli homogenates and endoglucanase 1 from culture filtrates of Trichoderma reesei in polyethylene glycol-phosphate systems. The goal was to test mathematical tools which could facilitate the optimisation procedure in aqueous two-phase systems. A modified simplex approach, the method of steepest ascent and a genetic algorithm were successfully applied and compared. The methods differ in the height of the optimum found, the number of experiments and the time required. The genetic algorithm proved to be an optimisation procedure which can be used well in aqueous two-phase systems. The simplex procedure has to be further improved.

Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems.

Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)4 tag. In contrast, the extraction yield of wild type cutinase was 2-3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.

Mary Parker Follett: epilogue to or return of a social work management pioneer?

The authors present an overview of the major concepts and contributions of Mary Parker Follett to the development of social work management theory, past and present. The case is made for Follett to be afforded a more visible role in the profession's understanding and teaching of modern management theory and practice. The authors show how Follett's work served as an important bridge between changing eras in management thought, specifically as related to scientific management and the subsequent human relations models. Even after six decades, Follett's work, in its timelessness and timeliness, remains contemporary, for it is rooted in her keen ability to grasp the complexities inherent in the human services enterprise, and thus it continues to inform one's understanding and behavior within the organizational context.