Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6‐anhydro‐β‐d ‐glucopyranose) in the presence of Mg²⁺–ATP. For the Lipomyces starkeyi LGK, we show here with real‐time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α‐d ‐glucose‐6‐phosphate in a manner reminiscent of an inverting β‐glycoside hydrolase. Kinetic characterization revealed the Mg²⁺ concentration for optimum activity (20–50 mm ), the apparent binding of levoglucosan (K_m=180 mm ) and ATP (K_m=1.0 mm ), as well as the inhibition by ADP (K_i=0.45 mm ) and d ‐glucose‐6‐phosphate (IC₅₀=56 mm ). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d ‐glucose‐6‐phosphate and ADP was observed under a broad range of conditions. 6‐Phospho‐α‐d ‐glucopyranosyl fluoride and 6‐phospho‐1,5‐anhydro‐2‐deoxy‐d ‐arabino‐hex‐1‐enitol (6‐phospho‐d ‐glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip ⁴C₁→¹C₄ required for 1,6‐anhydro‐product synthesis from d ‐glucose‐6‐phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.     

Mesoporous zeolite single crystal catalysts: Diffusion and catalysis in hierarchical zeolites

During the last years, several new routes to produce zeolites with controlled mesoporosity have appeared. Moreover, an improved catalytic performance of the resulting mesoporous zeolites over conventional zeolites has been demonstrated in several reactions. In most cases, the mesoporous zeolites exhibit higher catalytic activity, but in some cases also improved selectivity and longer catalyst lifetime has been reported. The beneficial effects of introducing mesopores into the zeolites has in most instances been attributed to improved mass transport to and from the active sites located in the zeolite micropores. Here, we briefly discuss the most important ways of introducing mesopores into zeolites and, for the first time, we show experimentally that the presence of mesopores dramatically increases the rate of diffusion in zeolite catalysts. This is done by studying the elution of iso-butane from packed beds of conventional and mesoporous zeolite catalysts. Moreover, we discuss in detail the recent observation of improved activity and selectivity in the alkylation of benzene with ethene using mesoporous zeolite single crystal catalysts. For this reaction, we show by calculation of the Thiele modulus that this improved performance can be mainly attributed to a diffusional limitation of ethylbenzene in the zeolite pores. This is verified in new ethylbenzene dealkylation experiments where mesoporous zeolite catalysts show significantly improved activity over conventional zeolite catalysts.     

Surface functionalization for self-referencing surface plasmon resonance (SPR) biosensors by multi-step self-assembly

Recently, a novel SPR sensor with on-chip referencing has been realized. In this sensor, one-half of the gold sensing surface is covered with a high refractive index overlayer of tantalum pentoxide (Ta₂O₅). When polychromatic beam illuminates the sensing surface, surface plasmon resonance in the areas with and without the overlayer occur at different wavelengths. Therefore, the reflected light exhibits two dips associated with SPRs in those two areas. When functionalized properly, one of the areas can be used as a specific sensing channel for detection of specific bio-interactions and the other can act as a reference channel for compensation for background refractive index fluctuations. In this paper we present a new functionalization approach for these mixed architecture chips. The gold side of the chip is functionalized with a mixed self-assembled monolayer of polyethylene oxide (PEO) and biotin terminated (BAT) thiols whereas the Ta₂O₅ side is coated with PEO terminated silanes. The PEO terminated thiols and silanes serve as a protein resistant background, while the biotin terminated thiols are used to bind streptavidin, which in turn immobilizes biotinylated antibodies. Hence, the gold side of the chip is used for the binding and detection of target analytes and the Ta₂O₅ side functions as a reference channel that monitors bulk refractive index changes and temperature drift. We have studied human chorionic gonadotropin (hCG) as a model system, currently detecting down to 5 ng/ml. In addition, we demonstrate the power of the on-chip reference channel for compensating for refractive index changes and eliminating false alarms.