Fumihiko Hasegawa

Transcriptional analysis of genes for energy catabolism and hydrolytic enzymes in the filamentous fungus Aspergillus oryzae using cDNA microarrays and expressed sequence tags

Aspergillus oryzae is a fungus used extensively in the fermentation industry. We constructed cDNA microarrays comprising 2,070 highly expressed cDNAs selected from the ∼6,000 non-redundant expressed sequence tags (ESTs) in the A. oryzae EST database (http://www.aist.go.jp/RIODB/ffdb/index.html). Using the cDNA microarrays, we analyzed the gene expression profiles of A. oryzae cells grown under the glucose-rich (AC) and glucose-depleted (AN) liquid culture conditions used during the construction of the EST database. The sets of genes identified by the cDNA microarray as highly expressed under each culture condition agreed well with the highly redundant ESTs obtained under the same conditions. In particular, transcription levels of most catabolic genes of the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle were higher under AC than AN conditions, suggesting that A. oryzae uses both EMP and TCA for glucose metabolism under AC conditions. We further studied the expression of genes encoding hydrolytic enzymes and enzymes involved in energy catabolism by using three industrial solid-phase biomass media, including wheat-bran. The wheat-bran culture gave the richest gene expression profile of hydrolytic enzymes and the lowest expression levels of catabolic genes (EMP, TCA) among the three media tested. The low expression levels of catabolic genes in the wheat-bran culture may release catabolite repression, consequently leading to the rich expression profiles of the hydrolytic enzymes.

Impact of Aspergillus oryzae genomics on industrial production of metabolites.

Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.

The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces

When fungi grow on plant or insect surfaces coated with wax polyesters that protect against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Aerial structures such as hyphae and conidiophores are coated with hydrophobins, which are surface‐active proteins involved in adhesion to hydrophobic surfaces. When the industrial fungus Aspergillus oryzae was cultivated in a liquid medium containing the biodegradable polyester polybutylene succinate‐coadipate (PBSA), the rolA gene encoding hydrophobin RolA was highly transcribed. High levels of RolA and its localization on the cell surface in the presence of PBSA were confirmed by immunostaining. Under these conditions, A. oryzae simultaneously produced the cutinase CutL1, which hydrolyses PBSA. Pre‐incubation of PBSA with RolA stimulated PBSA degradation by CutL1, suggesting that RolA bound to the PBSA surface was required for the stimulation. Immunostaining revealed that PBSA films coated with RolA specifically adsorbed CutL1. Quartz crystal microbalance analyses further demonstrated that RolA attached to a hydrophobic sensor chip specifically adsorbed CutL1. Circular dichroism spectra of soluble‐state RolA and bound RolA suggested that RolA underwent a conformational change after its adsorption to hydrophobic surfaces. These results suggest that RolA adsorbed to the hydrophobic surface of PBSA recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis. A fluorescence recovery after photobleaching experiment on PBSA films coated with FITC‐labelled RolA suggested that RolA moves laterally on the film. We discuss the novel molecular functions of RolA with regard to plastic degradation.

Novel hydrophobic surface binding protein, HsbA, produced by Aspergillus oryzae

ABSTRACT Hydrophobic surface binding protein A (HsbA) is a secreted protein (14.5 kDa) isolated from the culture broth of Aspergillus oryzae RIB40 grown in a medium containing polybutylene succinate-co-adipate (PBSA) as a sole carbon source. We purified HsbA from the culture broth and determined its N-terminal amino acid sequence. We found a DNA sequence encoding a protein whose N terminus matched that of purified HsbA in the A. ozyzae genomic sequence. We cloned the hsbA genomic DNA and cDNA from A. oryzae and constructed a recombinant A. oryzae strain highly expressing hsbA. Orthologues of HsbA were present in animal pathogenic and entomopathogenic fungi. Heterologously synthesized HsbA was purified and biochemically characterized. Although the HsbA amino acid sequence suggests that HsbA may be hydrophilic, HsbA adsorbed to hydrophobic PBSA surfaces in the presence of NaCl or CaCl2. When HsbA was adsorbed on the hydrophobic PBSA surfaces, it promoted PBSA degradation via the CutL1 polyesterase. CutL1 interacts directly with HsbA attached to the hydrophobic QCM electrode surface. These results suggest that when HsbA is adsorbed onto the PBSA surface, it recruits CutL1, and that when CutL1 is accumulated on the PBSA surface, it stimulates PBSA degradation. We previously reported that when the A. oryzae hydrophobin RolA is bound to PBSA surfaces, it too specifically recruits CutL1. Since HsbA is not a hydrophobin, A. oryzae may use several types of proteins to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation.

Chemistry of the M (M=Fe, Ca, Ba)-Se-H2O Systems at 25 °C

The chemistry of the M (M=Fe, Ca, Ba)-Se-H2O systems at 25 °C is reviewed based on our previous papers. In this paper, the phase equilibria in the Fe(III)-Se(IV)-H2O, Ca-Se(IV,VI)-H2O and Ba-Se(IV,VI)-H2O systems at 25 °C are discussed. Then, the three-stage process for removal of selenium from industrial waste water [Se(IV,VI) < 1,500 mg/L] containing sulfuric acid was introduced. This seems to be a promising process for selenium removal from acidic sulfate waste water containing high concentration levels of selenium to below 0.1 mg/L.

Amorphous titanium-oxide supercapacitors

The electric capacitance of an amorphous TiO2-x surface increases proportionally to the negative sixth power of the convex diameter d. This occurs because of the van der Waals attraction on the amorphous surface of up to 7 mF/cm2, accompanied by extreme enhanced electron trapping resulting from both the quantum-size effect and an offset effect from positive charges at oxygen-vacancy sites. Here we show that a supercapacitor, constructed with a distributed constant-equipment circuit of large resistance and small capacitance on the amorphous TiO2-x surface, illuminated a red LED for 37 ms after it was charged with 1 mA at 10 V. The fabricated device showed no dielectric breakdown up to 1,100 V. Based on this approach, further advances in the development of amorphous titanium-dioxide supercapacitors might be attained by integrating oxide ribbons with a micro-electro mechanical system.

Superior electric storage on an amorphous perfluorinated polymer surface.

Amorphous perfluoroalkenyl vinyl ether polymer devices can store a remarkably powerful electric charge because their surface contains nanometre-sized cavities that are sensitive to the so-called quantum-size effect. With a work function of approximately 10 eV, the devices show a near-vertical line in the Nyquist diagram and a horizontal line near the −90° phase angle in the Bode diagram. Moreover, they have an integrated effect on the surface area for constant current discharging. This effect can be explained by the distributed constant electric circuit with a parallel assembly of nanometre-sized capacitors on a highly insulating polymer. The device can illuminate a red LED light for 3 ms after charging it with 1 mA at 10 V. Further gains might be attained by integrating polymer sheets with a micro-electro mechanical system.

Amorphous aluminum-oxide supercapacitors

An amorphous aluminium-oxide supercapacitor can store a large amount of electric charge on its blackish, uneven Al2 O3−x surface, which has a convexity of 21 nm, AlO6 clusters, and a resistivity of . The electric storage can be elucidated by extremely enhanced electron trapping resulting from both the quantum-size effect and the offset effect of the positive charges at O-vacancy sites. Here, a supercapacitor, characterised by a high surface area of and the formation of large numbers of electric double layers, demonstrates a vertical line in the Nyquist diagram, corresponding to rapidly increased imaginary impedance and phase angle of with decreasing frequency in the Bode diagram using a distributed constant-electric circuit with a parallel assembly of nanometer-sized capacitors on a highly insulating amorphous aluminium surface. The supercapacitor indicates a switching effect for both positive and negative potentials. Thus, we can apply rechargeable dry solid supercapacitors in place of practical Li ions.

Realizing a supercapacitor in an electrical circuit

Capacitors are commonly used in electronic resonance circuits; however, capacitors have not been used for storing large amounts of electrical energy in electrical circuits. Here, we report a superior RC circuit which serves as an electrical storage system characterized by quick charging and long-term discharging of electricity. The improved energy storage characteristics in this mixed electric circuit (R1 + R2C1) with small resistor R1, large resistor R2, and large capacitor C1 are derived from the damming effect by large R2 in simple parallel R2C1 circuit. However, no research work has been carried out previously on the use of capacitors as electrical energy storage devices in circuits. Combined with nanotechnology, we hope that our finding will play a remarkable role in a variety of applications such as hybrid electric vehicles and backup power supplies.