| Pinoresinol diglucoside [(+)-1-pinoresinol 4,4′-di-O-β-D-glucopyranoside](PDG) is a major antihypertensive compound found in the bark of Tu-Chung(E. ulmoides Oliver), with great value in medicine. E. ulmoides Oliver is the major resource of PDG production, however, the produce period of PDG in E. ulmoides Oliver is long and the extraction process can only yield low amounts of product. Therefore it would have a good prospect for bioconversion PDG by microorganism. In this study, mung bean can promote the production of PDG by Phomopsis sp. XP-8; mung bean starch and mung bean water-soluble polysaccharide were found to be the main effective components and the monosaccharide components of mung bean water-soluble polysaccharide have different stimulative effect on the production of PDG and the related products, like [(+)-pinoresinol-4-O-β-Dglucopyranosi--de](PMG) and pinoresinol [(+)-pinoresinol](Pin). By analyzing the accumulation of the intermediate products and the changes of related enzyme activities during the bioconversion of PDG, PMG and Pin by Phomopsis sp. XP-8 using mung bean starch and mung bean water-soluble polysaccharide, we found that mung bean starch and mung bean water-soluble polysaccharide may be converted to phenylpropanoid pathway substrates which might promote the production of PDG and its related products. By precursors feeding method, stable isotope tracing and enzyme activity measurement, we analyzed the metabolic flow during the bioconversion of PDG, PMG and Pin, and found that the metabolic flow in Phomopsis sp. XP-8 was similar to the phenylpropanoid pathway in plants. In addition, after analyzing the actives of the products of Phomopsis sp. XP-8 resting cells, we found that the products had anti-cancer cell actives, and the main bioactive components were found to be PMG and Pin,so the related products of Phomopsis sp. XP-8 have potential application. The main content and results of study were as follows:(1) The production of PDG by Phomopsis sp. XP-8 in different natural mediumBased on the lignans biosynthesis pathway in plants, we selected the plant materials which are rich in precursors and substrates in the pathway, then measured the PDG production by Phomopsis sp. XP-8 when they were as the medium. The results showed that most of the plant materials could improve the production of PDG by Phomopsis sp. XP-8, and mung bean had the highest promoting effect. Furthermore, we optimized the cultured manner when used mung bean as the medium, and found that Phomopsis sp. XP-8 could produce more PDG production on the agar solid medium than that on the liquid medium; therefore, we choose to study the PDG yield on mung bean grain solid media by Phomopsis sp. XP-8 to adapt to industrial production. The highest PDG production was found to be 72.1 mg / kg(wet basis) or 216.3 mg / kg(dry basis) after 12 days fermentation on the mung bean grain medium containing 100 g/L glucose, which was much higher than that of the optimal synthesis medium in the preliminary. Meanwhile, PMG and Pin were also found in the fermentation medium by Phomopsis sp. XP-8. So it had a good potential in the production practice.(2) Analyzed the main components in mung beans which can improve the production of PDG by Phomopsis sp. XP-8To find out the main mechanism that mung bean could enhance the production of PDG by Phomopsis sp. XP-8, we analyzed the main components in mung beans,and studied the effect on the production of PDG of mung bean starch, water-soluble polysaccharide and protein in Phomopsis sp. XP-8 cell system respectively, with mung beans whole powder as control. The results showed that mung bean starch and mung bean water-soluble polysaccharide were the main effective components in mung bean which promoted the production of PDG by Phomopsis sp. XP-8. Mung bean starch was the main component that can improve the production of PDG and Pin; mung bean water-soluble polysaccharide contributed to the production of PMG and Pin. We found that glucose and arabinose could enhance the total production of the three products, and mostly contributed to the production of Pin, xylose and mannose can promote the production of PDG, while galactose promoted the production of PMG after analyzed the monosaccharide components of mung bean water-soluble polysaccharide. The results showed that, the promoting effect of mung bean on PDG production by Phomopsis sp. XP-8 mainly due to mung bean starch and water-soluble polysaccharide.(3) Research on the mechanism of the influence of mung bean components on the production of PDG and related products by Phomopsis sp. XP-8To study the mechanism of the promotive effect of mung bean starch and water-soluble polysaccharide on the bioconversion of PDG, PMG and/or Pin by Phomopsis sp. XP-8, we measured the type and accumulation of the intermediate products and the related enzyme activities during the bioconversion, evidences of the sequence accumulation of intermediate products and the related enzyme activities were similar to phenylpropanoid pathway in plants during the accumulation of PDG, PMG and/or Pin by Phomopsis sp. XP-8 using mung bean starch and water-soluble polysaccharide. So we speculated that mung bean starch and water-soluble polysaccharide might through improving the accumulation of the intermediate products in the related pathway fistly, then this intermediate products enhanced the production of PDG and related products.(4) Research on the metabolism flow of the bioconversion PDG and related products in Phomopsis sp. XP-8To find out the bioconversion pathway of PDG and the related products in Phomopsis sp. XP-8, we used precursor feeding method, metabolism flow analysing and stable isotope tracing. The results showed that the Phomopsis sp. XP-8 could biotransform the intermediate substances in phenylpropane pathway as the sole substrates to be Pin, then to be PDG with the addition of a small amount of glucose. The metabolism flow was:(phenylalanine, cinnamic acid)/ tyrosine, p-coumaric acid, Pin and/or PDG. 13 C labeled glucose and phenylalanine(with phenyl labeled) were used as the substrates to elucidate the metabolism flow in Phomopsis sp. XP-8. The results proved that the pathway from phenylalanine to the bioconversion of Pin in Phomopsis sp. XP-8, and the accumulation of cinnamic acid and p-coumaric acid was found during the bioconversion. The phenyl of phenylalanine participated in the bioconversion of the production. More over, glucose contributed to the bioconversion of phenylalanine and the flowing products. The result provided more evidences for that the bioconversion of PDG and related products in Phomopsis sp. XP-8 might through phenylpropane pathway.(5) Analyzed the anti-cancer activity of the products of Phomopsis sp. XP-8 resting cellsIn order to exploit the potential applications of the products of Phomopsis sp. XP-8 resting cells, inhibition effects on HepG-2 and K562 cells of the extract solution of Phomopsis sp. XP-8 resting cells were assessed. The result showed that the extract solution significantly inhibited cell viability and induced apoptosis on HepG-2 and K562 by up-regulating the expression of apoptosis related genes(FasL and caspase 3, 8, 9, 10); it also significantly inhibited HepG-2 cells adherence to the plate, and inhibited them from migrating by blocking the expression of MMP-9. The results indicated the products of Phomopsis sp. XP-8 had the potential as the anti-tumor drugs. |
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