The 26S proteasome is a 2.5 MDa macromolecular machine responsible for targeted protein degradation. Recently, four chaperones were identified that promote the assembly of the 19S regulatory particle (RP). Here, we probe the dynamic architecture of the proteasome by applying quantitative proteomics and mass spectrometry (MS) of intact complexes to provide a detailed characterization of how Ubp6 assists this assembly process. Our MS data demonstrate stoichiometric binding of chaperones and Ubp6 to the basal part of the RP. Genetic interactions of Ubp6 with Hsm3, but not with the other chaperones, indicate a functional overlay with Hsm3. Our biochemical data identified Ubp6 as an additional member of the Hsm3 module. Deletions of ubp6 with hsm3 perturb 26S proteasome assembly, which we attribute to an accumulation of ubiquitylated substrates on these assembly precursors. We therefore propose that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity.

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The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.

Removal of phenolic compounds from a raw industrial wastewater from phenolic resin processing, of which the components are phenol (8.9 wt%), m-and p-cresols (0.33 wt%), and xylenols (0.044 wt%), was carried out by using crosslinked cyclodextrin particles as a sorbent. A series of sorbents was prepared by varying the combination of cyclodextrin (CyD), β-CyD, γ -CyD, Mix-CyD (α-CyD:β-CyD:γ -CyD:dextrin = 30:10:10:50 wt/wt), the crosslinker, hexamethylene diisocyanate (HDI) or toluene-2,6-diisocyanate, and their molar ratio in the reaction batch. The removal of the phenolic compounds from raw industrial wastewater was an instantaneous process and was completed within about 5 min. The best removal efficiency was obtained by the crosslinked β-CyD with HDI in a 1:8 molar ratio or the crosslinked Mix-CyD with HDI, also in a 1:8 molar ratio. The prepared sorbents were efficiently regenerated by elution of the adsorbed phenols from the crosslinked polymers with methanol.

Glucose oxidase-containing liposomes (GOL) as well as detergent-modulated glucose oxidase-containing liposomes were prepared and characterized, focusing not only on the reactivity of the liposomes upon external addition of glucose but also on the leakage of the entrapped glucose oxidase (GO) from the liposomes with the aim of developing a reactive and stable liposomal GO system. The membranes of the GOL prepared were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modulated with either Triton X-100 or cholate. In the absence of added detergent, no GO leakage from the GOL was observed while its enzymatic activity was very low (low glucose permeability). As detergent-modulated liposomes, mixed POPC/Triton X-100 and mixed POPC/cholate liposomes (abbreviated as TL and CL, respectively) were prepared at different effective detergent/POPC molar ratios (R(e)) ranging from R(e) = 0 to R(e) = R(e) (sat) (R(e) (sat) is the critical value of R(e) at which the liposome membrane is saturated with detergent). The reactivity of GO-loaded TL (abbreviated as GOTL) or GO-loaded CL (GOCL) increased drastically with increase in the respective detergent content in the liposomes. In the case of GOTL, at R(e) (sat) = 0.40, a high reactivity was measured with a simultaneous high extent of GO leakage, suggesting that the observed enzymatic reaction was catalyzed mainly by leaked GO, caused by the interaction of Triton X-100 with the POPC membrane. On the other hand, GOCL prepared at R(e) (sat) = 0.43 showed relatively high reactivity with only a small extent of GO leakage, suggesting that most of the enzyme reaction was limited by the glucose permeation across the bilayers of GOCL. The GO leakage from GOCL was found to occur mostly during the rearrangement of the liposomal membrane during the preparation of the GOCL (mixing the GOL and cholate). Fluorescence polarization measurements of membrane-associated DPH (1,6-diphenyl-1,3,5-hexatriene) indicated that CL prepared by modifying POPC with cholate did not lead to a drastic change in membrane fluidity, indicating that the interacting cholate molecules did not penetrate deeply into the POPC bilayers. In summary, it was clearly shown that the membrane permeability of GOL can be quite simply modulated by mixing it with a certain amount of cholate to form highly reactive and stable GOCL with minimal enzyme leakage.

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