Characterization of earthworm α-amylases for dietary supplement development and biomass utilization.

Earthworms are useful soil-decomposing animals that possess various saccharification enzymes such as cellulases and amylases. Earthworms have also been traditionally used as antipyretic agents and medicines for preventing thrombotic diseases such as brain infarction. We previously developed a novel earthworm dietary supplement with fibrinolytic, cellulase, and amylase activities using high-pressure technology. However, the optimal temperature and pH required for amylase activity in bioindustry have not yet been investigated. In the present study, we purified and characterized two α-amylases of Eisenia fetida Waki, EfAMY1 and EfAMY2, which were monomeric enzymes of 63.8 kDa and 64.0 kDa, with specific activities of 69.2 and 40.4 units/mg, respectively. The optimal pH was 5.5 for both enzymes, and the optimal temperatures were 45 °C and 35 °C for EfAMY1 and EfAMY2, respectively; however, the enzymes were stable over a wide pH range (5-10) and at high temperature (up to 40 °C). These amylases showed higher specific activity and cold tolerance than those previously reported. These data should help to promote the development of E. fetida AMYs as functional dietary supplements and in biomass utilization.

High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.

Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), ß-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment.

An extract from brown rice inhibits signal transduction of angiotensin II in vascular smooth muscle cells.

BACKGROUND: Health benefits of brown rice over white rice have been described previously. However, whether the outer bran of rice contains an ingredient useful to prevent cardiovascular diseases remains unknown. The subaleurone layer of rice, which is usually lost by milling brown rice for whitening, is rich in varied nutrients, suggesting that some ingredient contained within this layer may be beneficial for the cardiovascular system. METHODS: To assess potential benefits of the subaleurone layer toward pathological vascular remodeling, we examined the effects of the layer extracts from Japanese rice (Oryza sativa var. japonica) on angiotensin II (Ang II)-induced signal transduction and hypertrophy in cultured rat vascular smooth muscle cells (VSMCs). RESULTS: Pretreatment of the ethyl acetate extract (100 µg/ml), but not other extracts, inhibited Ang II (100 nmol/l)-induced immediate signal transduction events. Also, the extract diminished c-Fos expression and hypertrophic protein accumulation induced by Ang II in the cells. CONCLUSION: These data suggest that an ingredient in the ethyl acetate extract from the subaleurone layer of rice has a protective effect toward cardiovascular diseases by interfering with signal transduction induced by Ang II.

Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase.

We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.