Tadashi Hatanaka

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), β-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), β-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC₅₀ = 11.41–23.44 mg/mL) and α-amylase (IC₅₀ = 18.07–53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption.

Streptomyces lividans is an efficient host for extracellular overproduction of recombinant proteins. To enhance the overexpression strength of S. lividans, we designed several kinds of expression plasmid with different positioning of repeat promoters. The effect of repeat promoters was evaluated by measuring the accumulated amounts of a stable transglutaminase or an unstable carboxypeptidase that was secreted into the medium. Successive tandem position of repeat promoter upstream the normal promoter did not enhance the expression of transglutaminase. Discrete position of repeat promoters both upstream and downstream the normal promoter enhanced the expression of transglutaminase to two-fold; and the downstream ones also enhanced the expression of carboxypeptidase to 1.7-fold. On the hand, there were still some constructs of plasmids with discrete repeat promoters that did not promote the expression of the target enzymes, indicating the complexity of the mechanisms of repeat promoters working on gene expression.

Streptomyces lividans is a potent host for the extracellular overproduction of heterologous proteins. To further improve the usability and productivity of S. lividans, a dual gene expression vector of “pTSKr duet” containing two strong constitutive promoters, scmpP_c and kasOp*, was constructed. The success in the overproduction of two secretary enzymes simultaneously without interference to each other indicated that the “pTSKr duet” vector can realize the coexpression of two genes simultaneously and independently. Further, using the two-gene coexpression vector, we screened the effects of the overexpression of five factors that possibly promoting secretion on the extracellular overproduction of heterologous secretory proteins. Interestingly, the coexpression of a quality control regulator (CssR) promoted the overproduction level to 1.3-fold for a stable heterologous protein of SMTG (transglutaminase from S. mobaraensis), while other four factors limited the overproduction of SMTG at different degrees.

We have previously reported a powerful promoter from the Streptomyces cinnamoneus TH-2 strain named ‘scmp’ and created an expression vector of pTONA5a for expression using S. lividans. The full-length scmp promoter sequence consists of 424 bp upstream of a metalloendoprotease gene in the S. cinnamoneus TH-2 genome. The promoter works in the presence of inorganic phosphate and glucose. In this study, we present the essential region of the scmp promoter (promoter C), which lacks 358 bp of the 5′ region of the full-length promoter. Promoter C was very short and contained only 63 bp. Using promoter C, we succeeded in the extracellular production of the Streptomyces enzymes of leucine aminopeptidase, ferulic acid esterase, and transglutaminase, which possessed signal peptides for secretion via the type II secretion pathway, at high levels.

We have previously reported a powerful promoter from the Streptomyces cinnamoneus TH-2 strain named ‘scmp’ and created an expression vector of pTONA5a based on pIJ702 for expression using S. lividans (Hatanaka et al. (2008) Protein Expr Purif 62: 244-248). The full-length scmp promoter sequence consists of 424 bp upstream of a metalloendoprotease gene in the S. cinnamoneus TH-2 genome. The promoter works in the presence of inorganic phosphate and glucose. In this study, we present the essential region of the scmp promoter (promoter C), which lacks 358 bp of the 5′ region of the full-length promoter. Promoter C was very short and contained only 63 bp. Moreover, there was no restriction on the medium component by combining the promoter and the S. livians 1326 strain. Using promoter C and S. lividans, we succeeded in the extracellular production of the Streptomyces enzymes leucine aminopeptidase, ferulic acid esterase, and transglutaminase, which possessed signal peptides for secretion via the second pathway, at high levels.

Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith belongs to the Alpinia genus in the Zingiberaceae family. In East Asia, Alpinia zerumbet has been widely used as food and traditional medicine. Previously, we identified proanthocyanidins (PACs), an anti-plant-virus molecule in A. zerumbet, using Nicotiana benthamiana and tomato mosaic virus (ToMV). Here, we found that PACs from A. zerumbet, apple, and green tea effectively inhibited ToMV infection. Additionally, the PACs from A. zerumbet exhibited greater antiviral activity than those from apple and green tea. The PACs from A. zerumbet also effectively inactivated influenza A virus and porcine epidemic diarrhea virus (PEDV), which acts as a surrogate for human coronaviruses, in a dose-dependent manner. The results from the cytopathic effect assays indicated that 0.1 mg/ml PACs from A. zerumbet decreased the titer of influenza A virus and PEDV by >3 log. These findings suggested that the direct treatment of viruses with PACs from A. zerumbet before inoculation reduced viral activity; thus, PACs might inhibit infections by an influenza virus, coronaviruses, and plant viruses.

Proanthocyanidins (PACs) have various bioactivities, such as being anti-bacterial, anti-cancer, and anti-oxidant. Consequently, they have been vigorously studied for the development of new natural bioactive compounds. Recently, AzPAC was isolated from the medicinal plant Alpinia zerumbet, and it was found to inhibit the infection of animal viruses, influenza A viruses (IAVs), and porcine epidemic diarrhea virus. The virucidal activity of AzPAC means that it can interact directly with viral particles. However, few studies have investigated the preventive mechanism utilized by AzPAC on influenza virus replication. In this study, the composition of AzPAC and the affinity between AzPAC and IAVs was investigated in detail. We found that AzPAC was composed of an epicatechin monomer, which was linked by inter-flavan bonds between the C4 and C8 positions (B2-type) and the C4 and C6 positions (B5-type) in the terminal units of the PAC. A quenching assay indicated that AzPAC interacted with IAV membrane proteins, hemagglutinin and neuraminidase. Additionally, circular dichroism analysis indicated that AzPAC affected the change in the secondary structure rate of the viral membrane proteins. AzPAC was able to impair the infective process of IAVs via direct interaction with their viral membrane proteins. These results indicate that A. zerumbet is an invaluable bioresource for the development of preventive drugs against IAV infection.

In plants, viral diseases are second only to fungal diseases in terms of occurrence, and cause substantial damage to agricultural crops. The aqueous extracts of shell ginger,Alpinia zerumbetexhibit inhibitory effects against virus infections in belonging to the Solanaceae family. In this study, we isolated an anti-plant-virus molecule from the extracts using a conventional method involving a combination of reversed phase column chromatography, dialysis, and lyophilization. The anti-plant-virus molecule was identified as proanthocyanidin, which mostly consisted of epicatechin and exhibited more than 40 degrees of polymerization.

In plants, viral diseases are second only to fungal diseases in terms of occurrence, and cause substantial damage to agricultural crops. The aqueous extracts of shell ginger, Alpinia zerumbet exhibit inhibitory effects against virus infections in belonging to the Solanaceae family. In this study, we isolated an anti-plant-virus molecule from the extracts using a conventional method involving a combination of reversed phase column chromatography, dialysis, and lyophilization. The anti-plant-virus molecule was identified as proanthocyanidin, which mostly consisted of epicatechin and exhibited more than 40 degrees of polymerization.