Biotransformation of nitro-polycyclic aromatic compounds by vegetable and fruit cell extracts

Biotransformation of nitro-polycyclic aromatic compounds by vegetable and fruit cell extracts 1 2 first-author 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:junyang@dlut.edu.cnqingyang@dlut.edu.cn †E-mail:junyang@dlut.edu.cnqingyang@dlut.edu.cn junyang@dlut.edu.cn 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:junyang@dlut.edu.cnqingyang@dlut.edu.cn †E-mail:junyang@dlut.edu.cnqingyang@dlut.edu.cn junyang@dlut.edu.cn School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China Extracts from various vegetables and fruits were investigated for their abilities to reduce nitro-polycyclic aromatic hydrocarbons (NPAHs). The extracts from grape and onion exhibited an interesting selectivity, yielding corresponding hydroxylamines or amines as major products under mild conditions of 30 °C and pH 7.0. Grape extracts reduced the 4-nitro-1,8-naphthalic anhydride with the highest conversion rate ( > 99%) and the highest ratio of hydroxylamine to amine (95:5). In contrast, the onion extracts reduced 4-nitro-1,8-naphthalic anhydride with a conversion rate of 94% and a ratio of hydroxylamine to amine of 8:92. The thiol-reducing agent, β-mercaptoethanol, and metal cations, Ca 2+ and Mg 2+ , greatly increased the reductive efficiency. This work provides an alternative strategy for biotransformation of nitro-polycyclic compounds. Biotransformation Nitro-polycyclic aromatic compounds Plant cell extracts Hydroxylamine Amine Introduction 1 Nitro-polycyclic aromatic hydrocarbons (NPAHs) are widely distributed environmental pollutants (Muck et al., 2002 2002 ; Haritash and Kaushik, 2009 2009 ), and are often resistant to degradation under natural conditions. Generally, nitroreduction is the first step of remediation of nitroaromatic pollutants. Nitroreduction increases the solubility of nitro compounds greatly in aqueous environment (Spain, 1995 1995 ), which facilitates microbial biodegradation. Nitroreduction products of NPAHs, including hydroxylamine or amine derivatives, are extensively used in pharmaceuticals and chemical industries (Tocher, 1997 1997 ). Bioreduction of NPAHs is a significant pathway that has shown partial success in practice (Ramos et al., 2005 2005 ). Despite biodegradation by a wide variety of bacteria, few microorganisms are found to be able to degrade NPAHs (Castelli et al., 2008 2008 ). Furthermore, the slow growth rate of the microbial community is another drawback of microbial nitroaromatic degradation, perhaps due to high toxicity and low solubility of nitro compounds (Marvin-Sikkema and de Bont, 1994 1994 ; Ramos et al., 2005 2005 ; Yoon et al., 2006 2006 ; Roldán et al., 2008 2008 ). In addition, the uncertainties associated with changes in the indigenous microbial community remain unpredictable (Dua et al., 2002 2002 ; Wu et al., 2008 2008 ). These restrictions prompt the exploration of non-microbial biodegradation methods (Parrish et al., 2004 2004 ). Plants are good choices in the removal of organics in environmental remediation (Macek et al., 2000 2000 ) and as biocatalysts in asymmetric reduction (Matsuda et al., 2009 2009 ). Several species of grass, legume, aquatic weeds, and transgenic plants have been shown to degrade parent polycyclic aromatic hydrocarbons (PAHs) (Abhilash et al., 2009 2009 ; Haritash and Kaushik, 2009 2009 ), whereas they have not yet been explored in NPAHs reduction. Compared with whole-plant system, plant cell extracts offer experimental advantages in plant biodegradation capabilities, toxicity tolerance, and shorter culture periods (Doran, 2009 2009 ). Nitroreduction mainly yields corresponding hydroxylamines and aromatic amines, both of which are chemicals used in manufacturing semiconductors, pharmaceuticals, and fine chemicals. However, hydroxylamine derivatives are seldom obtained because they are immediately reduced forwards to amines. In addition, to separate a mix of hydroxylamines and amines is difficult because they have very similar polarities. Therefore, to provide a high yielding chemoselective route to produce hydroxylamine or amine is of great significance. In this study, we investigated reduction of nitro-polycyclic aromatic compounds by cell extracts derived from various common fruits and vegetables. This work may provide an alternative strategy for bioreduction of nitroaromatic compounds. Materials and methods 1 Plants and agents 2 Fresh fruits and vegetables (Table 1 1 ), including grape ( Vitis vinifera L.), apple ( Malus pumila Mill.), banana ( Musa balbisiana Colla), cherry ( Prunus pseudocerasus Lindl.), pear ( Pyrus pyrifolia (Burm.) Nak.), strawberry ( Fragaria ananassa Duch.), orange ( Citus reticulate Blanco), jujube ( Zizyphus jujuba Mill.), potato ( Solanum tuberosum L.), plum ( Prunus spp.), scallion ( Allium fistulosum L. var. caespitosum), topinambur ( Hippophae Rhamnoides Linn.), onion ( Allium cepa L.), and garlic ( Allium saticum L.) were purchased from a local market at typical maturity period. All chemicals were of high-performance liquid chromatography (HPLC) grade and were purchased from Sigma and Fluka (USA). Nitroreduction: http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551084&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551091&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551088&type= The conversion rate [mean±standard deviation (SD) of triplicate tests] was determined by RP-HPLC, based on a 120-h reaction at 30 °C and pH 7.0 ND: not determined Entry Plant extract Conversion rate (%) b b ‒NHOH vs. NH 2 1 Grape ( Vitis vinifera L.) >99±0.86 95:5 2 Apple ( Malus pumila Mill.) 90±1.09 85:15 3 Banana ( Musa balbisiana Colla) >99±1.21 81:19 4 Cherry ( Prunus pseudocerasus Lindl . ) >99±0.97 80:20 5 Pear ( Pyrus pyrifolia (Burm.) Nak.) 93±2.17 79:21 6 Strawberry ( Fragaria ananassa Duch.) 91±3.01 65:35 7 Orange ( Citus reticulate Blanco) 91±0.74 60:40 8 Jujube ( Zizyphus jujuba Mill.) 92±2.65 45:55 9 Potato ( Solanum tuberosum L.) >99±0.66 26:74 10 Plum ( Prunus spp.) 90±1.34 21:79 11 Scallion ( Allium fistulosum L. var. caespitosum) 91±2.80 11:89 12 Topinambur ( Hippophae Rhamnoides Linn.) >99±1.08 10:90 13 Onion ( Allium cepa L.) 94±3.27 8:92 14 Garlic ( Allium saticum L.) 0 ND c c Plant extract preparation 2 Fresh, undamaged, firm fruits and vegetables were chosen and first cleaned with detergent, rinsed under running tap water, and then were surface disinfected by immersing in 70% ethanol for 3 min and in 5 g/L NaClO for 5 min followed by three rinses with sterile distilled water. A total of 15 g of fruit or vegetable was frozen with liquid nitrogen and then ground into a powder. The powder was dispersed in 100 ml buffer (10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 20 mmol/L NaCl, pH 7.0), and the mixture was placed on a shaker for 2 h followed by centrifugation at 5 000× g for 30 min. The supernatant obtained was filtered extract through a 0.22-μm filter and stored at −20 °C until used. Nitroreduction assay 2 All solutions and samples were kept at 4 °C (unless otherwise stated). A total of 10 ml of the extract was added to 0.1 ml of 20 mg/ml nitroaromatic compound dissolved in dimethylsulfoxyde (DMSO) and incubated at 30 °C on a shaker set at 200 r/min. The reaction was terminated by adding 20 ml of ethyl acetate. The organic solvent in the sample was removed by vacuum evaporation, and the conversion rate for nitroaromatic compound was determined by HPLC. Conversion rate determined by reverse phase HPLC 2 Reverse phase HPLC (RP-HPLC) analyses of substrates and products were performed using an Agilent 1100 system (Agilent, USA) coupled to a UV detector and a Kromasil C 18 column (4.6 mm×250 mm i.d., 5 μm, 80 Å). The column flow rate was maintained at 0.8 ml/min and the eluent was monitored at 254 nm ( A 254 ). The separation conditions and the retention time for the different compounds are summarized in Tables 1 1 and 2 2 . Kinetics 2 For kinetic studies, 4-nitro-1,8-naphthalic anhydride was chosen as the model substrate. A total of 2 ml of the reaction mixture was taken out for RP-HPLC analysis at different time points: 0, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, and 264 h. A first-order rate equation [ln( c / c 0 )=− k 1 / t , where c is substrate concentration, c 0 is substrate initial concentration, k 1 is velocity constant, and t is reaction time] was used to analyze the kinetic rates of nitroreduction, and the pseudo-first-order rate constant k 1 was determined. Factors affecting nitroreduction assay 2 To investigate the effects of metal cations on the nitroreduction, 5 mmol/L of Mg 2+ , Ca 2+ , Li + , Zn 2+ , Sr 2+ , Hg 2+ , Cu 2+ , Co 2+ , Al 3+ , Ni 2+ , Mn 2+ , and Fe 3+ were independently added to the reaction mixture. To investigate the effects of inorganic anions on the nitroreduction, 5 mmol/L of IO 4 − , N 3− , SO 4 2− , SO 3 2− , CL − , and S 2 O 3 2− were independently added to the reaction mixture. To investigate the effects of other reducing chemicals, 10 mmol/L of dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), and β-mercaptoethanol (β-ME) were independently added to the reaction mixture. In each of the above reaction systems, the mixture including filtered plant extract in buffer (10 mmol/L HEPES, 20 mmol/L NaCl, pH 7.0) without these above mentioned compounds, or buffer blank acted as a control. Each reaction was run in triplicate. The statistical analysis of paired t -test was used according to the software Origin 7.0. The effect of pH on nitroreduction was also studied using either 20 mmol/L sodium phosphate (for pH 6.0, 6.5, 7.0, and 7.5) or 20 mmol/L Tris-HCl (for pH 7.0, 7.5, 8.0, and 8.5). Results and discussion 1 Plants showing chemoselectivity 2 Nitroaromatic reduction usually proceeds through the formation of nitroso or hydroxylamine intermediates that are seldom detected due to their short lives. As shown in Table 1 1 , except garlic ( Allium saticum L.), which exhibited no nitroreduction activity, the extracts of all the other fruits and vegetables were able to effectively reduce 4-nitro-1,8-naphthalic anhydrides to their corresponding arylhydroxylamine, followed by arylamine products, with a conversion rate of > 90%. And these plants exhibited chemo-selectivity. Extracts isolated from grape ( Vitis vinifera L.), apple ( Malus pumila Mill.), banana ( Musa balbisiana Colla), cherry ( Prunus pseudocerasus Lindl.), pear ( Pyrus pyrifolia (Burm.) Nak.), strawberry ( Fragaria ananassa Duch.) and orange ( Citus reticulate Blanco) yielded hydroxylamines as major products after 120 h, while other plant extracts, such as onion ( Allium cepa L.), jujube ( Zizyphus jujuba Mill.), potato ( Solanum tuberosum L.), plum ( Prunus spp.), scallion ( Allium fistulosum L. var. caespitosum), and topinambur ( Hippophae Rhamnoides Linn.) yielded amine products. Among them, the grape extracts reduced the nitroaromatic compounds with the highest conversion rate ( > 99%) and the highest ratio of hydroxylamine to amine (95:5). In contrast, the onion extracts reduced 4-nitro-1,8-naphthalic anhydride with a conversion rate of 94% and the ratio of hydroxylamine to amine of 8:92. The nearly complete hydroxylamine or amine products by a highly chemoselective catalysis with grape or onion cells will provide an alternative production strategy and easier purification. Because grape cells exhibit potential applications in hydroxylamine production, the following investigations were set to understand grape cells-catalyzed nitroreduction. Kinetic properties of 4-nitro-1,8-naphthalic anhydride reduction 2 The reduction of 4-nitro-1,8-naphthalic anhydride by grape extracts exhibited a pseudo-first-order reaction with a rate constant of 0.010 h −1 and a half-life of 60 h. In the process of nitroreduction, the concentration of hydroxylamine reached the highest level when the reaction was carried out for 120 h (Fig. 1 1 ). After 264 h, all products were transformed into amine. Thus, the production of hydroxylamine or amine was time-dependent and these two products could be obtained easily through controlling the reaction time. (a) HPLC results for grape extracts reducing the nitro group. Peak 1: 4-hydroxylamino-1,8-naphthalic anhydride; Peak 2: 4-amino-1,8-naphthalic anhydride; Peak 3: 4-nitro-1,8-naphthalic anhydride. (b) Time-course study results for grape extracts reducing the nitro group. The results represent mean±SD of triplicate tests. The conversion rate was determined by RP-HPLC Factors affecting nitroreduction assay 2 Inorganic anions, such as chloride and sulfate, have also shown their effects on redox reactions (Lewis and Ray, 1984 1984 ). In this study, strong oxidized inorganic anion IO 4 − and electron transporting inhibitor N 3− inhibited the nitroreduction significantly, while others, including SO 4 2− , SO 3 2− , CL − , and S 2 O 3 2− , did not strongly affect this reduction (Fig. 2 2 ). The relative activity was compared with the control without these compounds. The results represent mean±SD of triplicate tests. * P< 0.05, significant differences according to paired t -tests The effects of other chemicals include thiol-reducing agents (DTT and β-ME), serine protease inhibitor (PMSF), and metal chelator (EDTA) on the reduction of NPAHs were investigated. As shown in Fig. 2 2 , the nitroreduction was dramatically increased in the presence of β-ME, but inhibited by EDTA. Active metals, such as magnesium, aluminum, and zinc, are usually good reducing agents (Laine and Cheng, 2007 2007 ). We investigated the effects of metal cations on reduction of NPAHs by grape extracts. As shown in Fig. 2 2 , the nitroreduction process was significantly increased by Ca 2+ and Mg 2+ , but inhibited by Hg 2+ , Co 2+ , Al 3+ , Cu 2+ , and Ni 2+ . As shown in Fig. 3 3 , the optimum pH for the nitroreduction catalyzed by grape extracts was 7.0. The conversion rate was kept above 50% between pH 6.0‒8.5. Compared with specific enzymes, such as nitroreductase from E. coli (Dai et al., 2009 2009 ), Rhodococcus sp. (Shen et al., 2009 2009 ), and other sources (Hallas and Alexander, 1983 1983 ; Schackmann and Muller, 1991 1991 ; Medina et al., 2004 2004 ; Teramoto et al., 2004 2004 ; van Aken, 2009 2009 ), with pH ranging between 6.5‒7.5, the plant extracts exhibited advantages over a wide range of reaction pH. The results represent mean±SD of triplicate tests. The conversion rate was determined by RP-HPLC Substrate spectra 2 Several nitropolycyclic aromatic compounds, including 3-nitro-1,8-naphthalic anhydride, 4-nitro-1,8-naphthalic anhydride, 3-nitrophthalimide, and 4-nitrophthalimide, were investigated for substrate spectra of grape extracts. They could be reduced to corresponding hydroxylamine or amine by grape extracts with very high conversion rate ( > 86%) and chemoselectivity (Table 2 2 ). The conversion rate (mean±SD of triplicate tests) was determined by RP-HPLC. Different letters in the same column indicate significant differences ( P< 0.05) according to paired t -tests Entry Substrate HPLC condition Retention time of ‒NHOH/‒NH 2 /‒NO 2 (min) Conversion rate (%) ‒NHOH:‒NH 2 1 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551112&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551120&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551116&type= 30%‒100% CH 3 OH, 60 min 19.5/21.2/26.8 >99±1.88A 94:6D 2 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551124&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551126&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551125&type= 30%‒100% CH 3 OH, 60 min 20.5/22.0/27.2 >99±3.07A 95:5D 3 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551127&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551130&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551128&type= 30%‒51% CH 3 OH, 18 min 6.5/7.7/14.3 93±2.19B 88:12E 4 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551132&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551139&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23551136&type= 30%‒60% CH 3 OH, 15 min 6.4/7.2/7.4 86±3.98C 83:17F Conclusions 1 Plant-based biotransformation exhibited advantages over a wide range of mild conditions. Under 30 °C, 200 r/min, and pH 7.0, nitro-polycyclic aromatic compounds were reduced into the corresponding hydroxylamine or amine products within 120 h in the presence of grape or onion extracts. The nearly complete hydroxylamine or amine products by a highly chemoselective catalysis with grape or onion cells will provide an easier preparation of high-value hydroxylamine or amine compounds in chemical industries. The identification of the key active components in the future will facilitate full understanding and potential applications in biotransformation of nitroaromatic compounds. References PC Abhilash , S Jamil , N Singh . Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics . Biotechnol Adv , 2009 . 27 ( 4 ): 474 - 488 . 19371778 DOI: 10.1016/j.biotechadv.2009.04.002 http://doi.org/10.1016/j.biotechadv.2009.04.002 . 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Leaching of contaminated leaves following uptake and phytoremediation of RDX, HMX, and TNT by poplar 10.1631/jzus.B1100254 Project supported by the National Basic Research Program (973) of China (No. 2010CB126102), the National Natural Science Foundation of China (No. 31070715), the National High-Tech R&D Program (863) of China (No. 2011AA10A204), the National Key Technology R&D Program of China (No. 2011BAE06B05), and the Fundamental Research Funds for the Central Universities (No. DUT10LK33), China 2011-08-08 2012-03-15 2011-10-27 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 13