The gene encoding beta-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75 degrees C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100 degrees C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105 degrees C, respectively. rBglA was active toward artificial (p-nitrophenyl beta-D: -glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl beta-D: -glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and beta-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.

Eukaryotic replication protein A (RPA) is a singlestranded(ss) DNA binding protein with multiple functions in DNA replication, repair, and genetic recombination. The 70-kDa subunit of eukaryotic RPA contains a conserved four cysteine-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Recently, we described a novel function for the zinc-finger motif in the regulation of human RPA's ssDNA binding activity through reduction-oxidation (redox). Here, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its RPA32 and/or RPA14 subunits. Yeast RPA requires a reducing agent, such as dithiothreitol (DTT), for its ssDNA binding activity. Also, under non-reducing conditions, its DNA binding activity decreases 20 fold. In contrast, the RPA70 subunit does not require DTT for its DNA binding activity and is not affected by the redox condition. These results suggest that all three subunits are required for the regulation of RPA's DNA binding activity through redox potential.

The efficiency of a frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) channel has been evaluated by varying the length of the inlet frit element. In a FI-AFlFFF channel, a high speed frit flow is introduced through the inlet frit to provide hydrodynamic relaxation of the incoming sample stream from the channel inlet. The experimental plate heights and peak recovery values are examined for three different FI-AFlFFF channels by varying field strengths and the channel membrane materials. It has been found that the length of the inlet frit element influences the performance of hydrodynamic relaxation, as well as peak recovery in the FI-AFlFFF channel system. Experimental plate height data show that the hydrodynamic relaxation itself occurs more efficiently when the length of inlet frit is longer. While an FI-AFlFFF channel having a longer inlet frit (4.2 cm long) gives a good relaxation, it shows less efficiency in considering peak recovery over a range of the field strengths examined in this study. A similar tendency is observed when channel membrane is varied. By considering the efficiency of hydrodynamic relaxation and sample recovery during elution, an FI-AFlFFF channel of an intermediate length of inlet frit (3.2 cm) is shown, from experiments, to be optimum.

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