Phomopsis sp. XP-8 (an endophytic fungus) was previously found to produce pinoresinol diglucoside (PDG), a major antihypertensive compound of Tu-Chung (the bark of Eucommia ulmoides Oliv.), which is widely used in Chinese traditional medicines. In the present study, two bioconversion systems were de...
Phomopsis sp. XP-8 (an endophytic fungus) was previously found to produce pinoresinol diglucoside (PDG), a major antihypertensive compound of Tu-Chung (the bark of Eucommia ulmoides Oliv.), which is widely used in Chinese traditional medicines. In the present study, two bioconversion systems were developed for the production of PDG in Tris-HCl buffer containing glucose and Phomopsis sp. XP-8 cells (both resting and freeze-dried). When other factors remained unchanged, the bioconversion time, glucose concentration, cell ages, cell dosage, pH, temperature, and stirring speed influenced PDG production in a similar and decreasing manner after an initial increase with increasing levels for each factor. Considering the simultaneous change of various factors, the optimal conditions for PDG production were established as 70 g/l cells (8-day-old), 14 g/l glucose, $28^{\circ}C$, pH 7.5, and 180 rpm for systems employing resting cells, and 3.87 g/l cells, 14.67 g/l glucose, $28^{\circ}C$, pH 7.5, and 180 rpm for systems employing freeze-dried cells. The systems employing freeze-dried cells showed lower peak PDG production ($110.28{\mu}g/l$), but at a much shorter time (12.65 h) compared with resting cells (23.62 mg/l, 91.5 h). The specific PDG production levels were 1.92 and $24{\mu}g$ per gram cells per gram glucose for freeze-dried cells and resting cells, respectively. Both systems indicated a new and potentially efficient way to produce PDG independent of microbial cell growth.
Phomopsis sp. XP-8 (an endophytic fungus) was previously found to produce pinoresinol diglucoside (PDG), a major antihypertensive compound of Tu-Chung (the bark of Eucommia ulmoides Oliv.), which is widely used in Chinese traditional medicines. In the present study, two bioconversion systems were developed for the production of PDG in Tris-HCl buffer containing glucose and Phomopsis sp. XP-8 cells (both resting and freeze-dried). When other factors remained unchanged, the bioconversion time, glucose concentration, cell ages, cell dosage, pH, temperature, and stirring speed influenced PDG production in a similar and decreasing manner after an initial increase with increasing levels for each factor. Considering the simultaneous change of various factors, the optimal conditions for PDG production were established as 70 g/l cells (8-day-old), 14 g/l glucose, $28^{\circ}C$, pH 7.5, and 180 rpm for systems employing resting cells, and 3.87 g/l cells, 14.67 g/l glucose, $28^{\circ}C$, pH 7.5, and 180 rpm for systems employing freeze-dried cells. The systems employing freeze-dried cells showed lower peak PDG production ($110.28{\mu}g/l$), but at a much shorter time (12.65 h) compared with resting cells (23.62 mg/l, 91.5 h). The specific PDG production levels were 1.92 and $24{\mu}g$ per gram cells per gram glucose for freeze-dried cells and resting cells, respectively. Both systems indicated a new and potentially efficient way to produce PDG independent of microbial cell growth.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
05). Thus, we concluded the obtained mathematical model to be accurate and reliable for predicting the PDG production.
제안 방법
The parameters and their levels are listed in Table 1. A set of 17 experiments was conducted on the factors and levels according to the Box-Behnken designs, displayed in Table 2. The software package Design Expert (8.0.5; Stat-Ease Inc., USA) was used to design the experiments as well as to conduct data analysis.
A fitted model was applied to study the effect of the three chosen variables on the response and to establish the optimum experimental conditions that contribute to the highest PDG yield. Response surfaces plots and contour plots were attained, using the fitted model by keeping the least effective independent variable at a constant value, while changing the other two variables.
These results agree with several reports [25, 48, 49]. In conclusion, this study revealed that PDG can be effectively synthesized by microbial cells, thus simplifying the reaction system and shortening the transformation time; furthermore, a novel and simple method was provided for the production of PDG. The immobilization of whole microbial cells has many potential advantages over fermentation with free cells.
The measurement of PDG production was conducted via HPLC in a Shimadzu Prominence LC-20A analytical HPLC system (Shimadzu, Japan). The HPLC device was equipped with a quaternary pump, a SIL-20AF automated sample injector, SPDM20A, a photodiode-array detector, a CTO-20A incubator, a Waters (USA) XTerra MS C18-column (4.
To optimize the conditions for PDG production, the levels of bioconversion time, glucose concentration, and cell age were further improved via Box-Behnken design and RSM analysis with central points of 72 h duration, 15 g/l glucose concentration, and an 8-day cell age, respectively. The process of data analysis was conducted through PROC RSREG (response surface), where a quadratic response surface regression model was established, yielding the optimum level of the corresponding factor. As a result, the quadratic regression equation of PDG production was obtained regarding the following three variables: bioconversion time, glucose concentration, and cell ages.
XP-8 to eliminate the limitation caused by cell growth. Therefore, the present study focused on the development of a bioconversion system for the production of PDG, employing prepared resting and freeze-dried cells. Following this procedure, pre-cultured cells were utilized as a catalyst and the bioconversion of PDG occurred in a pattern of an enzymatic reaction.
To optimize the conditions for PDG production, the levels of bioconversion time, glucose concentration, and cell age were further improved via Box-Behnken design and RSM analysis with central points of 72 h duration, 15 g/l glucose concentration, and an 8-day cell age, respectively. The process of data analysis was conducted through PROC RSREG (response surface), where a quadratic response surface regression model was established, yielding the optimum level of the corresponding factor.
Response surfaces plots and contour plots were attained, using the fitted model by keeping the least effective independent variable at a constant value, while changing the other two variables. To validate the estimated model, a confirmatory experiment was conducted, utilizing optimal reaction conditions.
대상 데이터
The parameters and their levels are shown in Table 3. A three-factor and three-level design with 17 individual points was selected for this study (Table 4).
The HPLC device was equipped with a quaternary pump, a SIL-20AF automated sample injector, SPDM20A, a photodiode-array detector, a CTO-20A incubator, a Waters (USA) XTerra MS C18-column (4.6 × 250 mm, 5 μm), and an LC Solution software operating system (Shimadzu).
The Phomopsis sp. XP-8 used in this study was obtained from the China Center for Type Culture Collection (Wuhan, China) (code: CCTCC M209291). It was cultured on a potato dextrose agar (containing 200 g potato, 20 g dextrose, 20 g agar, and 1,000 ml tap water) medium slant and stored at 4°C prior to usage.
데이터처리
3D, suggested that bioconversion time and cell dosage had a substantial interactive effect on PDG production. These results agreed with the significance of regression coefficients of the quadratic polynomial model (Table 6).
성능/효과
Solving the equations revealed the optimum transformation conditions at the bioconversion time of 91.82 h: glucose concentration of 14.11 g/l, and cell age of 8.31 days. Under the achieved optimal conditions, the highest PDG production was estimated to be 23.
19. Therefore, our results confirmed that the substance that exhibited a retention time identical to that of the standard PDG in the HPLC-MS analysis of the samples was indeed PDG.
후속연구
This may be due to the freeze-drying process that destroyed the activity of some enzymes, thus resulting in low PDG production, but that released enzymes from cells and increased the reaction speed and resulted in a shorter reaction time. However, further studies are still required to identify the relevant enzymes and specific mechanisms involved in these processes. The mentioned effects could be related to different functions of glucose in the two systems: in the system with the freezedried cells, glucose was used mainly as a precursor and for the production of other precursors that are required for the formation of PDG, such as ATP and CoA.
참고문헌 (50)
Luo LF, Wu WH, Zhou YJ, Yan J, Yang GP, Ouyang DS. 2010. Antihypertensive effect of Eucommia ulmoides Oliv. extracts in spontaneously hypertensive rats. J. Ethnopharmacol. 129: 238-243.
Xie LH, Akao T, Hamasaki K, Deyama T, Hattori M. 2003. Biotransformation of pinoresinol diglucoside to mammalian lignans by human intestinal microflora, and isolation of Enterococcus faecalis strain PDG-1 responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol. Chem. Pharm. Bull. 51: 508-515.
Wang CZ, Ma XQ, Yang DH, Guo ZR, Liu GR, Zhao GX, et al. 2010. Production of enterodiol from defatted flaxseeds through biotransformation by human intestinal bacteria. BMC Microbiol. 10: 115.
Lee SY, Kwon HK, Lee SM. 2011. SHINBARO, a new herbal medicine with multifunctional mechanism for joint disease: first therapeutic application for the treatment of osteoarthritis. Arch. Pharm. Res. 34: 1773-1777.
Lee AS, Ellman MB, Yan D, Kroin JS, Cole BJ, van Wijnen AJ, et al. 2013. A current review of molecular mechanisms regarding osteoarthritis and pain. Gene 527: 440-447.
Lee SM, Kim HJ, Ha YJ, Park YN, Lee SK, Park YB, et al. 2012. Targeted chemo-photothermal treatments of rheumatoid arthritis using gold half-shell multifunctional nanoparticles. ACS Nano 7: 50-57.
Hemmati S, Schmidt TJ, Fuss E. 2007. (+)-Pinoresinol/(-)-lariciresinol reductase from Linum perenne Himmelszelt involved in the biosynthesis of justicidin B. FEBS Lett. 581: 603-610.
Wang PC, Ran XH, Luo HR, Ma QY, Liu YQ, Zhou J, et al. 2013. Phenolic compounds from the roots of Valeriana officinalis var. latifolia. J. Braz. Chem. Soc. 24: 1544-1548.
Wang Q, Wang C, Zuo Y, Wang Z, Yang B, Kuang H. 2012. Compounds from the roots and rhizomes of Valeriana amurensis protect against neurotoxicity in PC12 cells. Molecules 17: 15013-15021.
Liu WJ, Wang LB. 2010. The lignans from Daphne giraldii Nitsche. Chinese J. Med. Chem. 4: 014.
Dong X, Yang C, Xu G, Cao S, Fu J, Lin L, et al. 2016. Chemical constituents from Daphne giraldii Nitsche and their contents simultaneous determination by HPLC. Evid. Based Complement. Alternat. Med. 2016: 9492368.
Wang JL, Liu EW, Zhang Y, Wang T, Han LF, Gao XM. 2012. Validation of a HPLC-tandem MS/MS method for pharmacokinetics study of (+)-pinoresinol-di- ${\beta}$ -D-glucopyranoside from Eucommia ulmoides Oliv extract in rats' plasma. J. Ethnopharmacol. 139: 337-342.
Liu E, Han L, Wang J, He W, Shang H, Gao X, et al. 2012. Eucommia ulmoides bark protects against renal injury in cadmium-challenged rats. J. Med. Food 15: 307-314.
Nam JW, Kim SY, Yoon T, Lee YJ, Kil YS, Lee YS, et al. 2013. Heat shock factor 1 inducers from the bark of Eucommia ulmoides as cytoprotective agents. Chem. Biodivers. 10: 1322-1327.
Huang RH, Xiang Y, Liu XZ, Zhang Y, Hu Z, Wang DC. 2002. Two novel antifungal peptides distinct with a fivedisulfide motif from the bark of Eucommia ulmoides Oliv. FEBS Lett. 521: 87-90.
Vermes B, Seligmann O, Wagner H. 1991. Synthesis of biologically active tetrahydro-furofuranlignan-(syringin, pinoresinol)-mono-and bis-glucosides. Phytochemistry 30: 3087-3089.
Jeong EJ, Seo H, Yang H, Kim J, Sung SH, Kim YC. 2012. Anti-inflammatory phenolics isolated from Juniperus rigida leaves and twigs in lipopolysaccharide-stimulated RAW264. 7 macrophage cells. J. Enzyme Inhib. Med. Chem. 27: 875-879.
Tschaplinski TJ, Standaert RF, Engle NL, Martin MZ, Sangha AK, Parks JM, et al. 2012. Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog. Biotechnol. Biofuels 5: 1.
Shi J, Liu C, Liu L, Yang B, Zhang Y. 2012. Structure identification and fermentation characteristics of pinoresinol diglucoside produced by Phomopsis sp. isolated from Eucommia ulmoides Oliv. Appl. Microbiol. Biotechnol. 93: 1475-1483.
Zhang Y, Shi J, Gao Z, Yangwu R, Jiang H, Che J, et al. 2015. Production of pinoresinol diglucoside, pinoresinol monoglucoside, and pinoresinol by Phomopsis sp. XP-8 using mung bean and its major components. Appl. Microbiol. Biotechnol. 99: 4629-4643.
Zhang Y, Shi J, Gao Z, Che J, Shao D, Liu Y. 2016. Comparison of pinoresinol diglucoside production by Phomopsis sp. XP-8 in different media and the characterisation and product profiles of the cultivation in mung bean. J. Sci. Food Agric. 96: 4015-4025.
Wang W, Shi J, Yang B. 2008. Optimization of conditions for production of pinoresinol diglucosideby a strain of Phoma sp. Trans. Chin. Soc. Agric. Eng. 24: 287-290.
Liu G, Xiao X, Jiang H, Mei C, Ding Y. 2013. Detection of pH variable in solid-state fermentation process by FT-NIR spectroscopy and BP-Adaboost. Jiangsu Daxue Xuebao 34: 574-578.
Wang L, Meselhy MR, Li Y, QIN G-W, Hattori M. 2000. Human intestinal bacteria capable of transforming secoisolariciresinol diglucoside to mammalian lignans, enterodiol and enterolactone. Chem. Pharm. Bull. 48: 1606-1610.
Heinonen S, Nurmi T, Liukkonen K, Poutanen K, Wahala K, Deyama T, et al. 2001. In vitro metabolism of plant lignans: new precursors of mammalian lignans enterolactone and enterodiol. J. Agric. Food Chem. 49: 3178-3186.
Xie LH, Ahn EM, Akao T, Abdel-Hafez AAM, Nakamura N, Hattori M. 2003. Transformation of arctiin to estrogenic and antiestrogenic substances by human intestinal bacteria. Chem. Pharm. Bull. 51: 378-384.
Grishko VV, Tarasova EV, Ivshina IB. 2013. Biotransformation of betulin to betulone by growing and resting cells of the actinobacterium Rhodococcus rhodochrous IEGM 66. Process Biochem. 48: 1640-1644.
Fan L, Dong Y, Xu T, Zhang H, Chen Q. 2013. Gastrodin production from p-2-hydroxybenzyl alcohol through biotransformation by cultured cells of Aspergillus foetidus and Penicillium cyclopium. Appl. Biochem. Biotechnol. 170: 138-148.
Mikhailova R, Sapunova L, Lobanok A, Yasenko M, Shishko ZF. 2000. Isoelectrophoretic characterization of extracellular polygalacturonases of various Aspergillus alliaceus strains. Microbiology 69: 162-166.
Fan Y, Yu Y, Jia X, Chen X, Shen Y. 2013. Cloning, expression and medium optimization of validamycin glycosyltransferase from Streptomyces hygroscopicus var. jinggangensis for the biotransformation of validoxylamine A to produce validamycin A using free resting cells. Bioresour. Technol. 131: 13-20.
Satake H, Ono E, Murata J. 2013. Recent advances in the metabolic engineering of lignan biosynthesis pathways for the production of transgenic plant-based foods and supplements. J. Agric. Food Chem. 61: 11721-11729.
Yang Y, Jin Z, Jin Q, Dong M. 2015. Isolation and fatty acid analysis of lipid-producing endophytic fungi from wild Chinese Torreya grandis. Microbiology 84: 710-716.
Li Y, Peng X, Chen H. 2013. Comparative characterization of proteins secreted by Neurospora sitophila in solid-state and submerged fermentation. J. Biosci. Bioeng. 116: 493-498.
Pandey A, Selvakumar P, Soccol CR, Nigam P. 1999. Solidstate fermentation for the production of industrial enzymes. Curr. Sci. 77: 149-162.
Singhania RR, Sukumaran RK, Patel AK, Larroche C, Pandey A. 2010. Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases. Enzyme Microb. Technol. 46: 541-549.
Tobimatsu Y, Davidson CL, Grabber JH, Ralph J. 2011. Fluorescence-tagged monolignols: synthesis, and application to studying in vitro lignification. Biomacromolecules 12: 1752-1761.
Liu C, Shi J, Zhou X, Yang B, Dou X. 2011. Isolation, identification and growth conditions of endophytic fungi of Eucommia ulmoides Oliv. for production of PDG. J. Northwest A. F. Univ. 39: 203-209.
Feng S, Gan Z, Zhai X, Fu P, Sun W. 2006. Content comparison of pinoresinol diglucoside in original and reborn bark of Eucommia ulmoides. J. Chin. Med. Mater. 29: 792-794.
Yao LN, Su YF, Yin ZY, Qin N, Li TX, Si CL, et al. 2010. A new phenolic glucoside and flavonoids from the bark of Eucommia ulmoides Oliv. Holzforschung 64: 571-575.
Golubev W, Kulakovskaya T, Shashkov A, Kulakovskaya E, Golubev N. 2008. Antifungal cellobiose lipid secreted by the epiphytic yeast Pseudozyma graminicola. Microbiology 77: 171-175.
Qi F, Jing T, Zhan Y. 2012. Characterization of endophytic fungi from Acer ginnala Maxim. in an artificial plantation: media effect and tissue-dependent variation. PLoS One 7: e46785.
Zhang Y, Shi J, Liu L, Gao Z, Che J, Shao D, Liu Y. 2015. Bioconversion of pinoresinol diglucoside and pinoresinol from substrates in the phenylpropanoid pathway by resting cells of Phomopsis sp. XP-8. PLoS One 10: e0137066.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.