Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.

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Anti-tumor vaccines are a relatively non-toxic alternative to conventional chemotherapeutic strategies to control breast cancer. Immunization with tumor-associated antigens (TAAs) triggers anti-tumor cytotoxic T lymphocytes (CTL), which can limit tumor progression. Here we report on the development and effectiveness of a TAA-based DNA vaccine encoding Mage-b1/2, the mouse homologue of the human MAGE-B1/2. As model system, we used immune competent Balb/c mice with syngeneic non-metastatic (64pT) or metastatic (4TO7cg) breast tumors. First, the presence of Mage-btranscripts in the 64pT and 4TO7cg breast tumors and metastases was demonstrated by RT-PCR, Southern blotting, and DNA sequencing. A DNA-based vaccine was developed from transcripts of one of the 64pT tumors, encoding the complete Mage-b1/2 protein, and subsequently tested for its preventive efficacy in both breast tumor models. Mice were immunized two times intramuscularly with the vaccine (pcDNA3.1-Mage-b1/2-V5), the control vector (pcDNA3.1-V5), or saline. Two weeks after the last immunization, the syngeneic 4TO7cg or 64pT tumor cell lines were injected in a mammary fat pad. Mice were monitored during the next 4 weeks for tumor formation, latency and size, and subsequently sacrificed for analysis. While the Mage-b1/2 vaccine had only a minor effect on the latency and growth of primary tumors, a significant and reproducible reduction in the number of 4TO7cg metastases was observed (vaccine versus control vector, p=0.0329; vaccine versus saline, p=0.0128). The observed protective efficacy of the Mage-b DNA vaccine correlated with high levels of vaccine-induced IFNgamma in spleen and lymph nodes upon re-stimulation in vitro. These results demonstrate the potential of TAA-based DNA vaccines in controlling metastatic disease in breast cancer patients.

In this paper we report on the development of a label-free low-volume (12.5 microL), high-throughput microplate calorimetric biosensor for fast ascorbic acid quantification in food and pharmaceutical products. The sensor is based on microplate differential calorimetry (MiDiCal) technology in which the heat generation, due to the exothermic reaction between ascorbic acid and ascorbate oxidase, is differentially monitored between two neighboring wells of an IC-built wafer. A severe discrepancy is found between expected and observed sensor readings. To investigate the underlying mechanisms of these findings a mathematical model, taking into account the biochemical reactions and diffusion properties of oxygen, ascorbic acid, and ascorbate oxidase, is developed. This model shows that oxygen depletion in the microliter reaction volumes, immediately after injection of sample (ascorbic acid) into the well, causes the enzymatic reaction to slow down. Calibration experiments show that the sensor's signal is linearly correlated to the area under the output versus time profile for the ascorbic acid concentration range from 2.4 to 350 mM with a limit of detection of 0.8 mM. Validation experiments on fruit juice samples, food supplements, and a pain reliever supplemented with ascorbic acid reveal that the designed method correlates well with HPLC reference measurements. The main advantages of the presented biosensor are the low analysis cost due to the low amounts of enzyme and reagents required and the possibility to integrate the device in fully automated laboratory analysis systems for high-throughput screening and analysis.

A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharides D-fucose, D-xylose, L-arabinose, and D-galactose, as well as on L-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor D-glucose, resulting in drastically increased K m values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis.