Microbial transformation of indole-3-acetonitrile to indole-3-acetamide ...
Process Biochemistry 41 (2006) 1746–1750 www.elsevier.com/locate/procbio
Microbial transformation of indole-3-acetonitrile to indole-3-acetamide by Nocardia sp. 108 Ya-Jun Wang a, Yu-Guo Zheng a,*, Jian-Ping Xue b, Yin-Chu Shen a,b a
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang Province 310014, PR China b Shanghai Pesticide Research Institute, Shanghai 200032, PR China Received 5 January 2006; received in revised form 16 March 2006; accepted 21 March 2006
Abstract The ability of Nocardia sp. 108 to hydrate indole-3-acetonitrile to indole-3-acetamide was identified. Cell culture showed that Nocardia sp. 108 acrylonitrile-hydrating activity reached its maximum, 1209.8 U/g DCW, after cultivated at 28 8C for 45 h. Under the biotransformation conditions of 28 8C and pH 7.5, the maximal indole-3-acetonitrile conversion ratio and indole-3-acetamide yield were 34.34 and 32.68% (w/w), respectively. Moreover, the Km and Vmax values for the indole-3-acetonitrile biotransformation catalyzed by Nocardia sp. 108 were 1.18 102 mol/l and 1.16 103 mol/l min. Ethanol supplemented to the reaction system dramatically enhanced indole-3-acetonitrile aqueous solubility. Furthermore, 4% (v/v) ethyl acetate raised the indole-3-acetonitrile bioconversion capacity 78%. # 2006 Elsevier Ltd. All rights reserved. Keywords: Indole-3-acetoamide; Indole-3-acetonitrile; Nitrile hydratase; Nocardia; Biotransformation
1. Introduction Organic nitrile compounds exist extensively in bacteria, fungi, plant and so on. Although nitriles are generally toxic, many microorganisms can assimilate them as carbon and/or nitrogen sources. There are two known metabolic pathways to convert nitriles to the corresponding carboxylic acids in microorganisms [1]. Nitrilase catalyzes the direct hydrolysis of nitrile into the corresponding carboxylic acid, while nitrile hydratase (NHase) catalyzes the hydration of nitrile to amide and amide is subsequently hydrolyzed into carboxylic acid by the action of amidase [1–4]. NHase is of industrially importance in the acrylamide, nicotinamide production [5]. Extensively researches on NHases from Bacillus, Microbacterium imperiale, Comamonas, Corynebacterium, Rhodococcus, Pseudonocardia and Pseudomonas have been performed [5–10]. It is well known that indole-3-acetic acid is a kind of plant growth hormone. The presence of indole-3-acetonitrile in developing, dormant and stratified Fraxinus excelsior seeds was confirmed [11]. Also, it was found that indole-3-acetoamide is a
* Corresponding author. Tel.: +86 571 88320614; fax: +86 571 88320630. E-mail address: [email protected] (Y.-G. Zheng). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.03.016
characteristic intermediate in indole-3-acetic acid synthesis from tryptophan by the tryptophan-2-monooxygenase pathway [12]. So far Agrobacterium tumefaciens, Bradyrhizobium japonicum USDA 110, Bradyrhizobium elkanii USDA 76 and two Bradyrhizobium sp. strains, BTA-1 and BGA-1, have been found to be able to produce indole-3-acetoamide [13,14]. In addition to a precursor of plant growth hormone IAA, IAM is also an important intermediate in organic synthesis. A series of N-substituted indole-3-acetamide derivatives displaying good blocking activity to a1-adrenoceptor can be developed to drugs for benign prostatic hyperplasia (BPH) [15]. Also, some indole3-acetamide derivatives can be developed new antioxidant compounds, which function as free-radical scavenger in body and are localized within the phospholipid bilayer of cell membranes to protect against biological lipid peroxidation [16]. Some microorganism strains, such as Nocardia sp. 108, isolated by Shanghai Pesticide Research Institute, have been identified and used successfully for the commercial production of acrylamide in China [17]. Although Nocardia N.C.I.B. 11215 and Norcardia globerula NHB-2 have been reported to have a relatively broad substrate specificity and a versatile nitrile-degrading activity [18,19], documents concerning indole-3-acetonitrile hydration by Norcardia are still not available. The goals of the present work are to investigate the
Y.-J. Wang et al. / Process Biochemistry 41 (2006) 1746–1750
feasibility of indole-3-acetoamide bioconversion by Nocardia sp. 108 and the possible improvements of organic solvent supplementation on indole-3-acetonitrile biotransformation as well.
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phosphate, indole-3-acetonitrile and Nocardia sp. 108 cells were kept in wellsealed Erlenmeyer flasks, maintained at 28 8C and lasted for 1 h. After the bioconversion, Nocardia sp. 108 was removed by centrifuged at 6700 g for 10 min. Supernatant was collected and stored at 4 8C till gas chromatograph analysis. For straightforward comparison, the relative residual NHase activity of Nocardia sp. 108 r (%) was applied and defined as follows:
2. Materials and methods r ð%Þ ¼
2.1. Preparation of Nocardia sp. 108 resting cells The strain of Nocardia sp. 108, isolated by Shanghai Pesticide Research Institute, was used. Nocardia sp. 108 was streaked on agar plates containing (g/ l): 10.0 glucose, 3.0 yeast extract, 1.0 NaCl, 0.2 K2HPO4, 0.2 MgSO4 and 20.0 agar. All media were sterilized by autoclaving at 121 8C for 20 min. A single colony was used to inoculate one 10 ml liquid seed medium in a 50 ml Erlenmeyer flask to prepare preinocula for further fermentation. Fermentation media consisted of (g/l): 20.0 glucose, 5.0 yeast extract, 7.5 urea, 0.75 monosodium glutamate, 0.5 K2HPO4, 0.5 KH2PO4, 0.2 MgSO4 and 1.0 CoCl2 and adjusted to pH 7.0. The preinocula were inoculated into fermentation media and cultivated aerobically at 28 8C until the maximal acrylonitrile-hydrating activity obtained. Each 20 ml broth was centrifuged at 6700 g for 10 min. The sediment was further washed twice with 0.1 mol/l, pH 7.5, phosphate buffer. Afterwards, collected cells were suspended into an approximate volume of 0.1 mol/l phosphate buffer (pH 7.5), and shaken gently for 1 min to prepare a homogeneous Nocardia sp. 108 suspension with final biomass concentration of about 1.5 g dry cell weight per liter.
2.2. Chemicals Acrylonitrile and acrylamide were purchased from Sigma–Aldrich (USA). Indole-3-acetonitrile and indole-3-acetamide were both of chemical reagent grade, supplied by Shanghai Pesticide Research Institute. Methanol, ethanol, octane, toluene, ethyl acetate and butyl acetate, of analytical grade, were commercially available. Yeast extract and glucose were commercially available.
2.3. pH and temperature dependence of acrylonitrile-hydrating activity of Nocardia sp. 108 pH-Dependence of NHase activity of Nocardia sp. 108 was performed by exposing their suspension mentioned above in different buffers (pH range of 4.0–12.0) at 30 8C for 1 h. After incubation, these treated cells were recovered by centrifugation at 6700 g for 10 min, followed by suspended into 0.1 mol/l, pH 7.5, phosphate buffer for further residual acrylonitrile-hydrating activity assay. The temperature dependence of acrylonitrile-hydrating activity was carried out in a similar way to that of pH. The Nocardia sp. 108 suspensions were exposed to 0.1 mol/l, pH 7.5, phosphate buffer at temperature range of 10–55 8C for 1 or 8 h.
E 100 Eorig
(1)
where E denotes the residual indole-3-acetonitrile-hydrating activity after organic solvent supplement and Eorig is the original indole-3-acetonitrilehydrating activity prior to organic solvent treatment.
2.6. Activity assay and chemical analysis NHase activities of Nocardia sp. 108 were chromatographically measured through nitrile biotransformation. One NHase unit (U) was defined as the amount of enzyme needed for 1 mmol acrylamide or indole-3-acetamide formation from corresponding nitriles at 28 8C in 0.1 mol/l, pH 7.5, phosphate buffer. After acrylonitrile bioconversion, reaction mixture was centrifuged at 8000 g for 10 min to remove cells, and supernatant containing acrylamide was further microfiltrated by 0.45 mm membrane. The resultant filtrate was directly quantified using a gas chromatograph GC-14C (Shimadzu, Japan) with a 1 m Shimadzu glass column packed with Porapak Q from Supleco (Bellefonte, PA, USA). Nitrogen was used as the carrier gas at flow rate of 30.0 ml/ min. The oven temperature was 85 8C with an injector and flame ionization detector temperature of 240 8C. The pretreatment of indole-3-acetamide conversion suspension is similar to that of acrylamide. The resultant filtrate containing indole-3-acetonitrile and indole-3-acetamide were analyzed using HPLC according to the method of Pollmann et al. [12].
3. Results and discussion 3.1. Nocardia sp. 108 growth and cellular activity alteration Firstly, we observed variation in biomass and the cellular activity during the normal growth of Nocardia sp. 108 at 28 8C, shown in Fig. 1. In the initial period, Nocardia sp. 108 biomass increases exponentially with no significant lag phase, which may be related to the similarity in media composition. After this
2.4. Nocardia sp. 108 enzymatic parameters for indole-3-acetonitrile The Nocardia sp. 108 suspension and indole-3-acetonitrile were firstly set in 28 8C water bath separately for 10 min. Then they were mixed, and indole-3acetonitrile bioconversion was carried out at 28 8C. Samples were taken for indole-3-acetonitrile, indole-3-acetamide determinations at appropriate interval. Based on the biotransformation data, enzymatic parameters for indole-3acetonitrile, Km and Vmax were further obtained by data regression.
2.5. Impacts of organic solvents on indole-3-acetonitrile biotransformation Methanol, ethanol, octane, toluene, ethyl acetate and butyl acetate were added into the Nocardia sp. 108 cell suspension mentioned above according to varying volumetric ratios. The resultant mixtures consisting of organic solvents,
Fig. 1. Growth curve of Nocardia sp. 108 and cellular catalytic activity changes during the culture in the fermentation broth: OD600 (*); NHase activity (~). NHase activity of Nocardia sp. 108 was measured using acrylonitrile as substrate.
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period, the biomass increases slightly and enters the stationary growth phase after 62 h. From Fig. 1, the specific acrylonitrilehydrating activity increases with time from 9 to 45 h, and peaks at 1209.8 U/g DCW. Henceforth, the acrylonitrile-hydrating activity subsides with elongated culture time. Therefore, we selected Nocardia sp. 108 cultivated for 45 h as the biocatalyst for the following investigation on indole-3-acetamide bioconversion. The dependences of pH and temperature on acrylonitrile hydration by Nocardia sp. 108 resting cells were examined. Acidic conditions of pH less than 5.0 have adverse impact on the cellular activity. The optimal acrylonitrile hydration condition for Nocardia sp. 108 is at 28 8C, pH 7.0–7.5. Additionally, indole-3-acetoamide bioconversion from indole3-acetonitrile catalyzed by Nocardia sp. 108 at 28 8C, pH 7.5, is feasible, and represented in Scheme 1. 3.2. Nocardia sp. 108 enzymatic parameters for indole-3acetonitrile Due to low aqueous solubility of indole-3-acetonitrile, roughly 1128.5 mg/l, indole-3-acetoamide bioconversion rate by Nocardia sp. 108, correlated to indole-3-acetonitrile concentration, can be expressed by the Michaelis–Menten kinetic model: v¼
Vmax ½S Km þ ½S
(3)
As shown in Eq. (3), ln([S0]/[S])/t is linear to ([S0] [S])/t. Therefore, a plot of ln([S0]/[S])/t versus ([S0] [S])/t can be used to regress two important enzymatic parameters, Michaelis constant Km and the maximal enzyme catalyzed reaction rate Vmax, as the plot displays Vmax/Km as the intercept and 1/Km as the slope. The bioconversion occurs at varying initial indole-3acetonitrile concentrations, from 481.7 to 1128.5 mg/l, catalyzed by Nocardia sp. 108 at 28 8C, pH 7.5, listed in Table 1. Indole-3-acetonitrile conversion ratio and the indole-3acetoamide yield are both indole-3-acetonitrile concentrationdependent. The maximal conversion ratio and yield are 34.34 and 32.68% (w/w), respectively. For hydration reactions, product yield are commonly larger than substrate conversion
Scheme 1. Indole-3-acetoamide biosynthesis by Nocardia sp. 108.
[S0] (mg/l)
[S] (mg/l)
[P] (mg/l)
Conversion ratio (%)
Yield (%)
1128.5 923.4 710.0 481.7
813.8 654.4 502.0 316.3
351.0 300.0 232.0 148.3
27.89 29.13 29.30 34.34
31.10 32.49 32.68 30.79
ratio. However, the actual indole-3-acetoamide yield is 30.79% (w/w) at low indole-3-acetonitrile concentration of 481.7 mg/l, which is unexpectedly lower than the measured indole-3acetonitrile conversion ratio of 34.34% (w/w), indicating that Nocardia sp. 108 might have an amidase activity and low indole-3-acetonitrile concentration of 481.7 mg/l produces a negligible influence on amidase activity while high concentration of indole-3-acetonitrile exhibit strong inhibition on amidase activity. A plot of ln([S0]/[S])/t versus ([S0] [S])/t is shown in Fig. 2. By fitting these enzymatic data in Eq. (3), Km and Vmax for indole-3-acetonitrile are obtained, which are 1.18 102 mol/l and 1.16 103 mol/l min, respectively. 3.3. Indole-3-acetonitrile-hydrating activity of Nocardia sp. 108 in monophasic system
(2)
Integrating Eq. (2), relationship between ln([S0]/[S])/t and ([S0] [S])/t is founded as follows: 1 ½S0 1 ð½S0 ½SÞ Vmax ln ¼ þ t ½S Km t Km
Table 1 Indole-3-acetonitrile bioconversion by Nocardia sp. 108 at 28 8C, pH 7.5, for 5 min
To raise indole-3-acetonitrile aqueous solubility and consequent mass transport, we conducted the Nocardia sp. 108 catalyzing indole-3-acetonitrile hydration in monophasic systems containing the water-miscible organic solvents, such as methanol and ethanol. Ethanol significantly enhances indole-3acetonitrile aqueous solubility, however, both ethanol and methanol are detrimental to NHase activity (Fig. 3). Possible dehydration and consequent denaturation by strong polar solvents contribute substantially to the reduced bioconversion performance. Compared to methanol, low ethanol concentrations of less than 20% (v/v) cause little effect on indole-3-acetonitrile-
Fig. 2. Plot of ln([S0]/S)/t versus [P]/t for indole-3-acetonitrile: bioconversion performed at 28 8C, pH 7.5: experimental values (*); fitted values (–).
Y.-J. Wang et al. / Process Biochemistry 41 (2006) 1746–1750
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Fig. 3. Impacts of alcohol supplements on indole-3-acetonitrile hydration catalyzed by Nocardia sp. 108 at 28 8C, pH 7.5: methanol (*); ethanol (&).
Fig. 5. Impacts of varying organic solvent supplements on Nocardia sp. 108 indole-3-acetonitrile-hydrating activity: ethyl acetate (~); toluene (*); butyl acetate (&); octane (").
hydrating activity. However, ethanol at concentration above 20% (v/v) dramatically deactivates cellular activity (Fig. 3). Time course of indole-3-acetonitrile-hydrating activity and cellular activities at varying amount of methanol reduction were also investigated (Fig. 4). The retained indole-3-acetonitrilehydrating activity of Nocardia sp. 108 in 15% (v/v) ethanol solution is approximately 45% after treated for 9 h, and is further reduced to 15% after 24 h. The cellular activity in 30% (v/v) ethanol is nearly undetectable after 5 h treatment.
As water is essential to enzymatic activity and survival of microbial cells as well, biocatalysis by Nocardia sp. 108 in aqueous–organic biphasic system rather than in organic system is further evaluated. It is well known that log P value of a
solvent is a useful, critical parameter to predict the behavior of enzymes in a biphasic system. Generally, the higher the log P value, the weaker toxicity to cell. Herein, we chose ethyl acetate, butyl acetate, octane and toluene with log P > 4 to evaluate the indole-3-acetonitrile biotransformation performance of Nocardia sp. 108 in aqueous–organic biphasic systems (Fig. 5). The presence of ethyl acetate, butyl acetate or toluene has a significant effect on of Nocardia sp. 108 indole-3acetonitrile-hydrating activity, while octane has negligible influence. Ethyl acetate supplementation drastically enhanced the bioconversion of indole-3-acetonitrile at the volumetric ratio range from 0 to 4% (v/v). Indole-3-acetonitrile hydration by Nocardia sp. 108 peaks at 4% (v/v) ethyl acetate, representing a 78% increase. Above 4% (v/v) ethyl acetate, however, the bioconversion ratio declines abruptly. Excessive lipophilic solvents partially distorted the phospholipid bilayer and cell membranes. The indole-3-acetonitrile bioconversions
Fig. 4. Time course of change in Nocardia sp. 108 indole-3-acetonitrilehydrating activity in the presence of ethanol: 15% (v/v) EtOH (~); 20% (v/ v) EtOH (&); 25% (v/v) EtOH (*); 30% (v/v) EtOH (!).
Fig. 6. Time course of indole-3-acetonitrile-hydrating activity decrease in the presence of ethyl acetate (&); toluene (~); butyl acetate (*).
3.4. Indole-3-acetonitrile-hydrating activity of Nocardia sp. 108 in aqueous–organic biphasic system
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in the presence of butyl acetate and toluene follow a pattern similar to that of ethyl acetate supplementation. Time course of residual indole-3-acetonitrile-hydrating activities of Nocardia sp. 108 in the presence of ethyl acetate, butyl acetate or toluene were further investigated, and the results are illustrated in Fig. 6. They all produce a sharp reduction in cellular activity. However, more than 82% activity is retained during the initial 3 h. After 24 h treatment, 70% activity is lost in the aqueous–toluene system; 85% is lost in the aqueous–butyl acetate system; 94% is lost in the aqueous–ethyl acetate system. 4. Conclusions The acrylonitrile-hydrating activity of Nocardia sp. 108 approaches its maximum, 1209.8 U/g DCW, after 45 h of cultivation at 28 8C. It exhibits high acrylonitrile-hydrating activity at 28 8C, pH 7.0–7.5. Results from indole-3-acetonitrile biotransformation trial indicate that Nocardia sp. 108 can hydrate indole-3-acetonitrile hydration to indole-3-acetamide. Additionally, Km and Vmax for the indole-3-acetonitrile biotransformation catalyzed by Nocardia sp. 108 are 1.18 102 mol/l and 1.16 103 mol/l min, respectively. Organic solvents influence indole-3-acetonitrile solubility, especially ethanol, while their influences on indole-3-acetonitrile transformation are species-dependent. As expected, methanol and ethanol are detrimental to indole-3-acetamideconverting capacity. Ethyl acetate has a significant effect on indole-3-acetonitrile biotransformation, as well as butyl acetate and toluene, while octane has a negligible effect. 4% (v/v) ethyl acetate markedly raises indole-3-acetonitrile biotransformation by 78%. Therefore, an ideal condition for indole-3-acetamide biosynthesis by Nocardia sp. 108 at 28 8C, pH 7.5, supplemented with 4% (v/v) ethyl acetate is proposed. Acknowledgements This work was financially supported by the national major basic research development program of China (no. 2003CB716005) and fund from the Science and Technology Department of Zhejiang Province of China (no. 2005C31021). References [1] Asano Y. Overview of screening for new microbial catalysts and their uses in organic synthesis—selection and optimization of biocatalysts. J Biotechnol 2002;94:65–72.
[2] Asano Y, Tani Y, Yamada H. A new enzyme ‘‘nitrile hydratase’’ which degrades acetonitrile in combination with amidase. Agric Biol Chem 1980;44:2251–2. [3] Watanabe I, Satoh Y, Enomoto K. Screening, isolation and taxonomical properties of microorganisms having acrylonitrile-hydrating activity. Agric Biol Chem 1987;51:3193–9. [4] Banerjee A, Sharma R, Banerjee UC. The nitrile degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 2002; 60:33–44. [5] Yamada H, Kobayashi M. Nitrile hydratase and its application to industrial production of acrylamide. Biosci Biotechnol Biochem 1996;60:1391–400. [6] Graham D, Pereira R, Barfield D, Cowan D. Nitrile biotransformations using free and immobilized cells of a thermophilic Bacillus spp. Enzyme Microb Technol 2000;26:368–73. [7] Saroja N, Shamala TR, Tharanathan RN. Biodegradation of starch-gpolyacrylonitrile, a packaging material, by Bacillus cereus. Process Biochem 2000;36:119–25. [8] Cantarella M, Cantarella L, Gallifuoco A, Frezzini R, Spera A, Alfani F. A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production. J Mol Catal B: Enzym 2004;29:105–13. [9] Effenberger F, Graef BW. Chemo- and enantioselective hydrolysis of nitriles and acid amides, respectively, with resting cells of Rhodococcus sp. C3II and Rhodococcus erythropolis MP50. J Biotechnol 1998; 60:165–74. [10] Ramakrishna C, Desai JD. Superiority of cobalt induced acrylonitrile hydratase of Arthrobacter spp IPCB-3 for conversion of acrylonitrile to acrylamide. Biotechnol Lett 1993;15:267–70. [11] Blake PS, Taylor JM, Finch-Savage WE. Identification of abscisic acid, indole-3-acetic acid, jasmonic acid, indole-3-acetonitrile, methyl jasmonate and gibberellins in developing, dormant and stratified seeds of ash (Fraxinus excelsior). Plant Growth Regul 2002; 37:119–25. [12] Pollmann S, Mu¨ller A, Piotrowski M, Weiler EW. Occurrence and formation of indole-3-acetamide in Arabidopsis thaliana. Planta 2002; 216:155–61. [13] Kobayashi M, Fujita T, Shimizu S. Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens. Appl Microbiol Biotechnol 1996;45:176–81. [14] Vega-Herna´ndez MC, Leo´n-Barrios M, Pe´rez-Galdona R. Indole-3-acetic acid production from indole-3-acetonitrile in Bradyrhizobium. Soil Biol Biochem 2002;34:665–8. [15] Wu BX, Li MY, Jiang ZZ, Xia L. Design, synthesis and 3D-QSAR study of N-substituted-3-indolyl-acetamide series as a1-adrenoceptor antagonists. Chin J Org Chem 2004;24:1587–94 [in Chinese]. ¨ lgen S, C [16] O ¸ oban T. Synthesis and antioxidant properties of novel Nsubstituted indole-2-carboxamide and indole-3-acetamide derivatives. Arch Pharm 2002;7:331–8. [17] Zhang YH, Fang RP, Shen YC. Studies on a strain producing nitrile hydratase. Ind Microbiol 1998;28:1–5 [in Chinese]. [18] Harper DB. Characterization of a nitrilase from Nocardia sp. (Rhodochrous group) N.C.I.B. 11215 using p-hydroxybenzonitrile as sole carbon source. Int J Biochem 1985;17:677–83. [19] Bhalla TC, Kumar H. Norcardia globerula NHB-2: a versatile nitriledegrading organism. Can J Microbiol 2005;51:705–8.
Microbial transformation of indole-3-acetonitrile to indole-3-acetamide by Nocardia sp. 108 Ya-Jun Wang a, Yu-Guo Zheng a,*, Jian-Ping Xue b, Yin-Chu Shen a,b a
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang Province 310014, PR China b Shanghai Pesticide Research Institute, Shanghai 200032, PR China Received 5 January 2006; received in revised form 16 March 2006; accepted 21 March 2006
Abstract The ability of Nocardia sp. 108 to hydrate indole-3-acetonitrile to indole-3-acetamide was identified. Cell culture showed that Nocardia sp. 108 acrylonitrile-hydrating activity reached its maximum, 1209.8 U/g DCW, after cultivated at 28 8C for 45 h. Under the biotransformation conditions of 28 8C and pH 7.5, the maximal indole-3-acetonitrile conversion ratio and indole-3-acetamide yield were 34.34 and 32.68% (w/w), respectively. Moreover, the Km and Vmax values for the indole-3-acetonitrile biotransformation catalyzed by Nocardia sp. 108 were 1.18 102 mol/l and 1.16 103 mol/l min. Ethanol supplemented to the reaction system dramatically enhanced indole-3-acetonitrile aqueous solubility. Furthermore, 4% (v/v) ethyl acetate raised the indole-3-acetonitrile bioconversion capacity 78%. # 2006 Elsevier Ltd. All rights reserved. Keywords: Indole-3-acetoamide; Indole-3-acetonitrile; Nitrile hydratase; Nocardia; Biotransformation
1. Introduction Organic nitrile compounds exist extensively in bacteria, fungi, plant and so on. Although nitriles are generally toxic, many microorganisms can assimilate them as carbon and/or nitrogen sources. There are two known metabolic pathways to convert nitriles to the corresponding carboxylic acids in microorganisms [1]. Nitrilase catalyzes the direct hydrolysis of nitrile into the corresponding carboxylic acid, while nitrile hydratase (NHase) catalyzes the hydration of nitrile to amide and amide is subsequently hydrolyzed into carboxylic acid by the action of amidase [1–4]. NHase is of industrially importance in the acrylamide, nicotinamide production [5]. Extensively researches on NHases from Bacillus, Microbacterium imperiale, Comamonas, Corynebacterium, Rhodococcus, Pseudonocardia and Pseudomonas have been performed [5–10]. It is well known that indole-3-acetic acid is a kind of plant growth hormone. The presence of indole-3-acetonitrile in developing, dormant and stratified Fraxinus excelsior seeds was confirmed [11]. Also, it was found that indole-3-acetoamide is a
* Corresponding author. Tel.: +86 571 88320614; fax: +86 571 88320630. E-mail address: [email protected] (Y.-G. Zheng). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.03.016
characteristic intermediate in indole-3-acetic acid synthesis from tryptophan by the tryptophan-2-monooxygenase pathway [12]. So far Agrobacterium tumefaciens, Bradyrhizobium japonicum USDA 110, Bradyrhizobium elkanii USDA 76 and two Bradyrhizobium sp. strains, BTA-1 and BGA-1, have been found to be able to produce indole-3-acetoamide [13,14]. In addition to a precursor of plant growth hormone IAA, IAM is also an important intermediate in organic synthesis. A series of N-substituted indole-3-acetamide derivatives displaying good blocking activity to a1-adrenoceptor can be developed to drugs for benign prostatic hyperplasia (BPH) [15]. Also, some indole3-acetamide derivatives can be developed new antioxidant compounds, which function as free-radical scavenger in body and are localized within the phospholipid bilayer of cell membranes to protect against biological lipid peroxidation [16]. Some microorganism strains, such as Nocardia sp. 108, isolated by Shanghai Pesticide Research Institute, have been identified and used successfully for the commercial production of acrylamide in China [17]. Although Nocardia N.C.I.B. 11215 and Norcardia globerula NHB-2 have been reported to have a relatively broad substrate specificity and a versatile nitrile-degrading activity [18,19], documents concerning indole-3-acetonitrile hydration by Norcardia are still not available. The goals of the present work are to investigate the
Y.-J. Wang et al. / Process Biochemistry 41 (2006) 1746–1750
feasibility of indole-3-acetoamide bioconversion by Nocardia sp. 108 and the possible improvements of organic solvent supplementation on indole-3-acetonitrile biotransformation as well.
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phosphate, indole-3-acetonitrile and Nocardia sp. 108 cells were kept in wellsealed Erlenmeyer flasks, maintained at 28 8C and lasted for 1 h. After the bioconversion, Nocardia sp. 108 was removed by centrifuged at 6700 g for 10 min. Supernatant was collected and stored at 4 8C till gas chromatograph analysis. For straightforward comparison, the relative residual NHase activity of Nocardia sp. 108 r (%) was applied and defined as follows:
2. Materials and methods r ð%Þ ¼
2.1. Preparation of Nocardia sp. 108 resting cells The strain of Nocardia sp. 108, isolated by Shanghai Pesticide Research Institute, was used. Nocardia sp. 108 was streaked on agar plates containing (g/ l): 10.0 glucose, 3.0 yeast extract, 1.0 NaCl, 0.2 K2HPO4, 0.2 MgSO4 and 20.0 agar. All media were sterilized by autoclaving at 121 8C for 20 min. A single colony was used to inoculate one 10 ml liquid seed medium in a 50 ml Erlenmeyer flask to prepare preinocula for further fermentation. Fermentation media consisted of (g/l): 20.0 glucose, 5.0 yeast extract, 7.5 urea, 0.75 monosodium glutamate, 0.5 K2HPO4, 0.5 KH2PO4, 0.2 MgSO4 and 1.0 CoCl2 and adjusted to pH 7.0. The preinocula were inoculated into fermentation media and cultivated aerobically at 28 8C until the maximal acrylonitrile-hydrating activity obtained. Each 20 ml broth was centrifuged at 6700 g for 10 min. The sediment was further washed twice with 0.1 mol/l, pH 7.5, phosphate buffer. Afterwards, collected cells were suspended into an approximate volume of 0.1 mol/l phosphate buffer (pH 7.5), and shaken gently for 1 min to prepare a homogeneous Nocardia sp. 108 suspension with final biomass concentration of about 1.5 g dry cell weight per liter.
2.2. Chemicals Acrylonitrile and acrylamide were purchased from Sigma–Aldrich (USA). Indole-3-acetonitrile and indole-3-acetamide were both of chemical reagent grade, supplied by Shanghai Pesticide Research Institute. Methanol, ethanol, octane, toluene, ethyl acetate and butyl acetate, of analytical grade, were commercially available. Yeast extract and glucose were commercially available.
2.3. pH and temperature dependence of acrylonitrile-hydrating activity of Nocardia sp. 108 pH-Dependence of NHase activity of Nocardia sp. 108 was performed by exposing their suspension mentioned above in different buffers (pH range of 4.0–12.0) at 30 8C for 1 h. After incubation, these treated cells were recovered by centrifugation at 6700 g for 10 min, followed by suspended into 0.1 mol/l, pH 7.5, phosphate buffer for further residual acrylonitrile-hydrating activity assay. The temperature dependence of acrylonitrile-hydrating activity was carried out in a similar way to that of pH. The Nocardia sp. 108 suspensions were exposed to 0.1 mol/l, pH 7.5, phosphate buffer at temperature range of 10–55 8C for 1 or 8 h.
E 100 Eorig
(1)
where E denotes the residual indole-3-acetonitrile-hydrating activity after organic solvent supplement and Eorig is the original indole-3-acetonitrilehydrating activity prior to organic solvent treatment.
2.6. Activity assay and chemical analysis NHase activities of Nocardia sp. 108 were chromatographically measured through nitrile biotransformation. One NHase unit (U) was defined as the amount of enzyme needed for 1 mmol acrylamide or indole-3-acetamide formation from corresponding nitriles at 28 8C in 0.1 mol/l, pH 7.5, phosphate buffer. After acrylonitrile bioconversion, reaction mixture was centrifuged at 8000 g for 10 min to remove cells, and supernatant containing acrylamide was further microfiltrated by 0.45 mm membrane. The resultant filtrate was directly quantified using a gas chromatograph GC-14C (Shimadzu, Japan) with a 1 m Shimadzu glass column packed with Porapak Q from Supleco (Bellefonte, PA, USA). Nitrogen was used as the carrier gas at flow rate of 30.0 ml/ min. The oven temperature was 85 8C with an injector and flame ionization detector temperature of 240 8C. The pretreatment of indole-3-acetamide conversion suspension is similar to that of acrylamide. The resultant filtrate containing indole-3-acetonitrile and indole-3-acetamide were analyzed using HPLC according to the method of Pollmann et al. [12].
3. Results and discussion 3.1. Nocardia sp. 108 growth and cellular activity alteration Firstly, we observed variation in biomass and the cellular activity during the normal growth of Nocardia sp. 108 at 28 8C, shown in Fig. 1. In the initial period, Nocardia sp. 108 biomass increases exponentially with no significant lag phase, which may be related to the similarity in media composition. After this
2.4. Nocardia sp. 108 enzymatic parameters for indole-3-acetonitrile The Nocardia sp. 108 suspension and indole-3-acetonitrile were firstly set in 28 8C water bath separately for 10 min. Then they were mixed, and indole-3acetonitrile bioconversion was carried out at 28 8C. Samples were taken for indole-3-acetonitrile, indole-3-acetamide determinations at appropriate interval. Based on the biotransformation data, enzymatic parameters for indole-3acetonitrile, Km and Vmax were further obtained by data regression.
2.5. Impacts of organic solvents on indole-3-acetonitrile biotransformation Methanol, ethanol, octane, toluene, ethyl acetate and butyl acetate were added into the Nocardia sp. 108 cell suspension mentioned above according to varying volumetric ratios. The resultant mixtures consisting of organic solvents,
Fig. 1. Growth curve of Nocardia sp. 108 and cellular catalytic activity changes during the culture in the fermentation broth: OD600 (*); NHase activity (~). NHase activity of Nocardia sp. 108 was measured using acrylonitrile as substrate.
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period, the biomass increases slightly and enters the stationary growth phase after 62 h. From Fig. 1, the specific acrylonitrilehydrating activity increases with time from 9 to 45 h, and peaks at 1209.8 U/g DCW. Henceforth, the acrylonitrile-hydrating activity subsides with elongated culture time. Therefore, we selected Nocardia sp. 108 cultivated for 45 h as the biocatalyst for the following investigation on indole-3-acetamide bioconversion. The dependences of pH and temperature on acrylonitrile hydration by Nocardia sp. 108 resting cells were examined. Acidic conditions of pH less than 5.0 have adverse impact on the cellular activity. The optimal acrylonitrile hydration condition for Nocardia sp. 108 is at 28 8C, pH 7.0–7.5. Additionally, indole-3-acetoamide bioconversion from indole3-acetonitrile catalyzed by Nocardia sp. 108 at 28 8C, pH 7.5, is feasible, and represented in Scheme 1. 3.2. Nocardia sp. 108 enzymatic parameters for indole-3acetonitrile Due to low aqueous solubility of indole-3-acetonitrile, roughly 1128.5 mg/l, indole-3-acetoamide bioconversion rate by Nocardia sp. 108, correlated to indole-3-acetonitrile concentration, can be expressed by the Michaelis–Menten kinetic model: v¼
Vmax ½S Km þ ½S
(3)
As shown in Eq. (3), ln([S0]/[S])/t is linear to ([S0] [S])/t. Therefore, a plot of ln([S0]/[S])/t versus ([S0] [S])/t can be used to regress two important enzymatic parameters, Michaelis constant Km and the maximal enzyme catalyzed reaction rate Vmax, as the plot displays Vmax/Km as the intercept and 1/Km as the slope. The bioconversion occurs at varying initial indole-3acetonitrile concentrations, from 481.7 to 1128.5 mg/l, catalyzed by Nocardia sp. 108 at 28 8C, pH 7.5, listed in Table 1. Indole-3-acetonitrile conversion ratio and the indole-3acetoamide yield are both indole-3-acetonitrile concentrationdependent. The maximal conversion ratio and yield are 34.34 and 32.68% (w/w), respectively. For hydration reactions, product yield are commonly larger than substrate conversion
Scheme 1. Indole-3-acetoamide biosynthesis by Nocardia sp. 108.
[S0] (mg/l)
[S] (mg/l)
[P] (mg/l)
Conversion ratio (%)
Yield (%)
1128.5 923.4 710.0 481.7
813.8 654.4 502.0 316.3
351.0 300.0 232.0 148.3
27.89 29.13 29.30 34.34
31.10 32.49 32.68 30.79
ratio. However, the actual indole-3-acetoamide yield is 30.79% (w/w) at low indole-3-acetonitrile concentration of 481.7 mg/l, which is unexpectedly lower than the measured indole-3acetonitrile conversion ratio of 34.34% (w/w), indicating that Nocardia sp. 108 might have an amidase activity and low indole-3-acetonitrile concentration of 481.7 mg/l produces a negligible influence on amidase activity while high concentration of indole-3-acetonitrile exhibit strong inhibition on amidase activity. A plot of ln([S0]/[S])/t versus ([S0] [S])/t is shown in Fig. 2. By fitting these enzymatic data in Eq. (3), Km and Vmax for indole-3-acetonitrile are obtained, which are 1.18 102 mol/l and 1.16 103 mol/l min, respectively. 3.3. Indole-3-acetonitrile-hydrating activity of Nocardia sp. 108 in monophasic system
(2)
Integrating Eq. (2), relationship between ln([S0]/[S])/t and ([S0] [S])/t is founded as follows: 1 ½S0 1 ð½S0 ½SÞ Vmax ln ¼ þ t ½S Km t Km
Table 1 Indole-3-acetonitrile bioconversion by Nocardia sp. 108 at 28 8C, pH 7.5, for 5 min
To raise indole-3-acetonitrile aqueous solubility and consequent mass transport, we conducted the Nocardia sp. 108 catalyzing indole-3-acetonitrile hydration in monophasic systems containing the water-miscible organic solvents, such as methanol and ethanol. Ethanol significantly enhances indole-3acetonitrile aqueous solubility, however, both ethanol and methanol are detrimental to NHase activity (Fig. 3). Possible dehydration and consequent denaturation by strong polar solvents contribute substantially to the reduced bioconversion performance. Compared to methanol, low ethanol concentrations of less than 20% (v/v) cause little effect on indole-3-acetonitrile-
Fig. 2. Plot of ln([S0]/S)/t versus [P]/t for indole-3-acetonitrile: bioconversion performed at 28 8C, pH 7.5: experimental values (*); fitted values (–).
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Fig. 3. Impacts of alcohol supplements on indole-3-acetonitrile hydration catalyzed by Nocardia sp. 108 at 28 8C, pH 7.5: methanol (*); ethanol (&).
Fig. 5. Impacts of varying organic solvent supplements on Nocardia sp. 108 indole-3-acetonitrile-hydrating activity: ethyl acetate (~); toluene (*); butyl acetate (&); octane (").
hydrating activity. However, ethanol at concentration above 20% (v/v) dramatically deactivates cellular activity (Fig. 3). Time course of indole-3-acetonitrile-hydrating activity and cellular activities at varying amount of methanol reduction were also investigated (Fig. 4). The retained indole-3-acetonitrilehydrating activity of Nocardia sp. 108 in 15% (v/v) ethanol solution is approximately 45% after treated for 9 h, and is further reduced to 15% after 24 h. The cellular activity in 30% (v/v) ethanol is nearly undetectable after 5 h treatment.
As water is essential to enzymatic activity and survival of microbial cells as well, biocatalysis by Nocardia sp. 108 in aqueous–organic biphasic system rather than in organic system is further evaluated. It is well known that log P value of a
solvent is a useful, critical parameter to predict the behavior of enzymes in a biphasic system. Generally, the higher the log P value, the weaker toxicity to cell. Herein, we chose ethyl acetate, butyl acetate, octane and toluene with log P > 4 to evaluate the indole-3-acetonitrile biotransformation performance of Nocardia sp. 108 in aqueous–organic biphasic systems (Fig. 5). The presence of ethyl acetate, butyl acetate or toluene has a significant effect on of Nocardia sp. 108 indole-3acetonitrile-hydrating activity, while octane has negligible influence. Ethyl acetate supplementation drastically enhanced the bioconversion of indole-3-acetonitrile at the volumetric ratio range from 0 to 4% (v/v). Indole-3-acetonitrile hydration by Nocardia sp. 108 peaks at 4% (v/v) ethyl acetate, representing a 78% increase. Above 4% (v/v) ethyl acetate, however, the bioconversion ratio declines abruptly. Excessive lipophilic solvents partially distorted the phospholipid bilayer and cell membranes. The indole-3-acetonitrile bioconversions
Fig. 4. Time course of change in Nocardia sp. 108 indole-3-acetonitrilehydrating activity in the presence of ethanol: 15% (v/v) EtOH (~); 20% (v/ v) EtOH (&); 25% (v/v) EtOH (*); 30% (v/v) EtOH (!).
Fig. 6. Time course of indole-3-acetonitrile-hydrating activity decrease in the presence of ethyl acetate (&); toluene (~); butyl acetate (*).
3.4. Indole-3-acetonitrile-hydrating activity of Nocardia sp. 108 in aqueous–organic biphasic system
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in the presence of butyl acetate and toluene follow a pattern similar to that of ethyl acetate supplementation. Time course of residual indole-3-acetonitrile-hydrating activities of Nocardia sp. 108 in the presence of ethyl acetate, butyl acetate or toluene were further investigated, and the results are illustrated in Fig. 6. They all produce a sharp reduction in cellular activity. However, more than 82% activity is retained during the initial 3 h. After 24 h treatment, 70% activity is lost in the aqueous–toluene system; 85% is lost in the aqueous–butyl acetate system; 94% is lost in the aqueous–ethyl acetate system. 4. Conclusions The acrylonitrile-hydrating activity of Nocardia sp. 108 approaches its maximum, 1209.8 U/g DCW, after 45 h of cultivation at 28 8C. It exhibits high acrylonitrile-hydrating activity at 28 8C, pH 7.0–7.5. Results from indole-3-acetonitrile biotransformation trial indicate that Nocardia sp. 108 can hydrate indole-3-acetonitrile hydration to indole-3-acetamide. Additionally, Km and Vmax for the indole-3-acetonitrile biotransformation catalyzed by Nocardia sp. 108 are 1.18 102 mol/l and 1.16 103 mol/l min, respectively. Organic solvents influence indole-3-acetonitrile solubility, especially ethanol, while their influences on indole-3-acetonitrile transformation are species-dependent. As expected, methanol and ethanol are detrimental to indole-3-acetamideconverting capacity. Ethyl acetate has a significant effect on indole-3-acetonitrile biotransformation, as well as butyl acetate and toluene, while octane has a negligible effect. 4% (v/v) ethyl acetate markedly raises indole-3-acetonitrile biotransformation by 78%. Therefore, an ideal condition for indole-3-acetamide biosynthesis by Nocardia sp. 108 at 28 8C, pH 7.5, supplemented with 4% (v/v) ethyl acetate is proposed. Acknowledgements This work was financially supported by the national major basic research development program of China (no. 2003CB716005) and fund from the Science and Technology Department of Zhejiang Province of China (no. 2005C31021). References [1] Asano Y. Overview of screening for new microbial catalysts and their uses in organic synthesis—selection and optimization of biocatalysts. J Biotechnol 2002;94:65–72.
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