We first present a simple yet versatile strategy for the functionalization of polymer nanotubes in a controlled fashion. Carboxylic-acid-functionalized polypyrrole (CPPy) nanotubes were fabricated by using cylindrical micelle templates in a water-in-oil emulsion system, and the functional carboxyl groups were effectively incorporated into the polymer backbone during the polymerization by using pyrrole-3-carboxylic acid (P3CA) as a co-monomer without a sophisticated functionalization process. It was noteworthy that the chemical functionality of CPPy nanotubes was readily controlled in both qualitative and quantitative aspects. On the basis of the controlled functionality of CPPy nanotubes, a field-effect transistor (FET) sensor platform was constructed to detect specific biological entities by using a buffer solution as a liquid-ion gate. The CPPy nanotubes were covalently immobilized onto the microelectrode substrate to make a good electrical contact with the metal electrodes, and thrombin aptamers were bonded to the nanotube surface via covalent linkages as the molecular recognition element. The selective recognition ability of thrombin aptamers combined with the charge transport property of CPPy nanotubes enabled the direct and label-free electrical detection of thrombin proteins. Upon exposure to thrombin, the CPPy nanotube FET sensors showed a decrease in current flow, which was probably attributed to the dipole-dipole or dipole-charge interaction between thrombin proteins and the aptamer-conjugated polymer chains. Importantly, the sensor response was tuned by adjusting the chemical functionality of CPPy nanotubes. The efficacy of CPPy nanotube FET sensors was also demonstrated in human blood serum; this suggests that they may be used for practical diagnosis applications after further optimization.

We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-l-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications.

Recombinant tyrosinase from Streptomyces avermitilis MA4680, MelC2 (gi:499291317), was heterologously expressed in Escherichia coli BL21 (DE3). The expression level of active MelC2 was increased by the codon-optimized MelC1 caddie protein (KP198295.1). By performing saturation mutagenesis of the Y91 residue of MelC1, it was found that aromatic residues such as Y, F, and W at the 91st position help produce a correctly folded conformation of MelC2. The recombinant MelC2 was utilized as a biocatalyst to convert trans-resveratrol into piceatannol. In order to improve the product yield through suppression of the formation of melanin, a by-product, an increase in the ratio of monooxygenation (k 1) to dioxygenation (k 2) of MelC2 is desirable. This was achieved by a combination of protein engineering and regeneration of NADH with glucose dehydrogenase (GDH). Saturation mutagenesis was performed at 15 residues within 8-Å radius from copper ions of MelC2. A total of 2760 mutants were examined (99.7 % probability for NNK codon) and I41Y, a mutant, was screened. The ratio of k 1 to k 2 of the mutant increased sevenfold on tyrosine and fivefold on resveratrol, when compared to wild-type MelC2. As a result, the overall product yield from 500 μM resveratrol in 50-mL reaction was 15.4 % (77.4 μM piceatannol), 1.7 times higher than wild type. When I41Y was incorporated with the NADH regeneration system, the total product yield was 58.0 %, an eightfold increase (290.2 μM of piceatannol).

Regiospecific 3'-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K(m) and k(cat) values of CYP105D7 for daidzein were 21.83 +/- 6.3 microM and 15.01 +/- 0.6 min(-1) in the presence of 1 microM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3'-hydroxylation reaction of daidzein was carried out with 100 microM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6-fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3',4'-trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h.

We present a thin membrane transducer (TMT) that can detect nucleic acid based biomolecular reactions including DNA hybridization and protein recognition by aptamers. Specific molecular interactions on an extremely thin and flexible membrane surface cause the deflection of the membrane due to surface stress change which can be measured by a compact capacitive circuit. A gold-coated thin PDMS membrane assembled with metal patterned glass substrate is used to realize the capacitive detection. It is demonstrated that perfect match and mismatch hybridizations can be sharply discriminated with a 16-mer DNA oligonucleotide immobilized on the gold-coated surface. While the mismatched sample caused little capacitance change, the perfectly matched sample caused a well-defined capacitance decrease vs. time due to an upward deformation of the membrane by a compressive surface stress. Additionally, the TMT demonstrated the single nucleotide polymorphism (SNP) capabilities which enabled a detection of mismatching base pairs in the middle of the sequence. It is intriguing that the increase of capacitance, therefore a downward deflection due to tensile stress, was observed with the internal double mismatch hybridization. We further present the detection of thrombin protein through ligand-receptor type recognition with 15-mer thrombin aptamer as a receptor. Key aspects of this detection such as the effect of concentration variation are investigated. This capacitive thin membrane transducer presents a completely new approach for detecting biomolecular reactions with high sensitivity and specificity without molecular labelling and optical measurement.

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In the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galβ1,4Glc (3'-SL) and Neu5Ac(α2,6)Galβ1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Pasteurella multocida α2,3-sialyltransferase (α2,3PST) forms a by-product to a certain extent, owing to its multifunctional activity at pH below 7.0, and Photobacterium damselae α2,6-sialyltransferase (α2,6PdST) shows relatively low ST activity. In this study, α2,3PST and α2,6PdST were successfully engineered using a hybrid approach that combines rational design with site-saturation mutagenesis. Narrowly focused on the substrate-binding pocket of the STs, putative functional residues were selected by multiple sequence alignment and alanine scanning, and subsequently subjected to site-saturation mutagenesis. In the case of α2,3PST, R313N single mutation improved its activity slightly (by a factor of 1.5), and further improvement was obtained by making the double mutants (R313N/T265S and R313H/T265S) resulting in an overall 2-fold improvement in its specific α2,3 ST activity, which is mainly caused by the increase in kcat. It was revealed that the R313 mutations to N, D, Y, H or T greatly reduced the α2,6 ST side-reaction activity of α2,3PST at below pH 7.0. In the case of α2,6PdST, single-mutation L433S/T and double-mutation I411T/L433T exhibited 3- and 5-fold enhancement of the α2,6 ST-specific activity compared with the wild-type, respectively, via increase in kcat values. Our results show a very good model system for enhancing ST activity and demonstrate that the generated mutants could be used efficiently for the mass production of 3'-SL and 6'-SL with enhanced productivity and yield.

Secreted tyrosinase from melanin-forming Streptomyces avermitilis MA4680 was involved in both ortho-hydroxylation and further oxidation of trans-resveratrol, leading to the formation of melanin. This finding was confirmed by constructing deletion mutants of melC(2) and melD(2) encoding extracellular and intracellular tyrosinase, respectively; the melC2 deletion mutant did not produce piceatannol as well as melanin, whereas the melD2 deletion mutant oxidized resveratrol and synthesized melanin with the same yields, suggesting that MelC2 is responsible for ortho-hydroxylation of resveratrol. Extracellular tyrosinase (MelC2) efficiently converted trans-resveratrol into piceatannol in the presence of either tyrosinase inhibitors or reducing agents such as catechol, NADH, and ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific hydroxylation of trans-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of trans-resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of trans-resveratrol was dependent upon inhibitor concentration. Using 1.8 mg of wild-type Streptomyces avermitilis cells, the conversion yield of 100 μM trans-resveratrol to piceatannol was 78% in 3 h in the presence of 1 mM catechol, indicating 14 μM piceatannol h(-1) DCW mg(-1) specific productivity, which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases.