Biotransformation of Chloro-Substituted Indolesto Indigoids by Phenol Hydroxylasefrom Arthrobacter sp. W1

Shengnan Shi & Fang Ma & Tieheng Sun & Ang Li &Jiti Zhou & Yuanyuan Qu

Received: 26 January 2013 /Accepted: 9 April 2013 /Published online: 30 April 2013# Springer Science+Business Media New York 2013

Abstract Recombinant Escherichia coli cells expressing phenol hydroxylase (designated asstrain PHIND) were used to synthesize chloro-substituted indigoids by the transformation ofindoles. The optimal conditions for the biotransformation of 4- and 7-chloroindole weredetermined by response surface methodology. Biotransformation kinetic assays revealed thatstrain PHIND showed high catalytic efficiency for 4- and 7-chloroindole. The formation rateof 7,7′-dichloroindigo (1.35 unit/mg cell dry weight) by strain PHIND was 1.14-fold higherthan that of 4,4′-dichloroindigo. The intermediates of 7-chloroindole biotransformation wereidentified by high-performance liquid chromatography–mass spectroscopy, and the biotrans-formation mechanism was also proposed. These results suggested that there was a potentialapplication of strain PHIND in the biotransformation of chloro-substituted indoles to valuableindigoids.

Keywords Phenol hydroxylase . Chloro-substituted indoles . Biotransformation . Responsesurface methodology

Introduction

Indigo and its derivatives as useful dye stuffs and drugs are not only used for printing anddyeing but also possess the therapeutic values for a number of diseases, includingAlzheimer's disease, certain forms of cancer, and delayed hypersensitivity [1–6]. At present,

Appl Biochem Biotechnol (2013) 170:951–961DOI 10.1007/s12010-013-0234-y

S. Shi : F. Ma (*) : T. Sun :A. LiState Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology,Harbin 150090, Chinae-mail: hit_dlut@126.com

J. Zhou : Y. Qu (*)Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education),School of Environmental Science and Technology, Dalian University of Technology,Dalian 116024, Chinae-mail: qyy@dlut.edu.cn

many researchers are interested in the biotransformation of indole to indigo, and a number ofoxygenases have been shown to oxidize indole, leading ultimately to the production of theindustrially useful dye indigo via a combination of enzymatic and nonenzymatic reactions[7–11]. However, much work has been done to improve and optimize the biologicalproduction of indigo. Relatively few studies have been focused on the biosynthesis of indigoderivatives from substituted indoles, even though some of the indigo derivatives may alsoserve as dyes and pharmaceutical precursors. Meantime, manufacture of indigo derivativesby chemical routes is not desirable, due to the use of aggressive reagents, expensive andcomplicated starting materials, and energy-intensive and low yields. Therefore, it is neces-sary to investigate the biosynthesis of indigo derivatives from substituted indoles.

Among the substituted indoles, chloro-substituted indoles have attracted much attentionowing to lower toxicity than the bromo-substituted indoles, better solubility in water thanmethyl-substituted indoles, and relatively inert nature of the substituent [8]. It was showedthat chloro-substituted indoles could be used as a high-throughput strategy for screeningmutants in the solid phase and also be used to investigate the biotransformation capability ofoxygenases [10–13]. For example, Zhang et al. used 4-chloroindole as the substrate in the“indigo assay” for considerably improved pigment formation and the search for enhancedmutants of P450 2A6 [10]. Naphthalene dioxygenase, toluene dioxygenase, and phenolhydroxylases (mPHKL28 and mPHKL33) that transformed the chloro-substituted indolesshowed that none of them could transform all kinds of chloro-substituted indoles [12,13].Naphthalene dioxygenase and mPHKL28 could catalyze 5-, 6-, and 7-chloroindole. Incontrast, toluene dioxygenase showed a limited activity towards 6- and 7-chloroindole,and mPHKL33 could not catalyze chloro-substituted indoles to indigoids [12,13]. However,there is a limited report on phenol hydroxylase from Arthrobacter sp. strains as a biocatalystfor chloro-substituted indigoids from indoles [14]. Thus, the use of phenol hydroxylase inthe area of biocatalysis chloro-substituted indigoids needs to be thoroughly exploited.

In this work, phenol hydroxylase from Arthrobacter sp. W1 was used to oxidize 4- and 7-chloroindoles to indigoids. The biotransformation conditions were optimized by responsesurface methodology (RSM). Biotransformation kinetic assays were utilized to analyze thecatalytic activity of strain PHIND. The intermediates generated from 7-chloroindole were alsoidentified by high-performance liquid chromatography–mass spectroscopy (HPLC-MS).Our work should exhibit the potential application of phenol hydroxylase in the biosynthesisof indigoids.

Materials and Methods

Chemicals, Bacterial Strain, and Culture Condition

The 4- and 7-chloroindole, kanamycin, isopropyl 1-thio-β-D-galactopyranoside (IPTG), andCH3OH were purchased from J&K Chemical Company (Beijing, China). All other reagentsand solvents were obtained from general commercial suppliers and used without furtherpurification. Escherichia coli BL21 (DE3) expressing phenol hydroxylase was used in thisstudy, which was designed as strain PHIND. Strain PHIND was grown in LB medium with30 μg/mL kanamycin as a selective pressure to an optical density at 600 nm (OD600) of 0.4at 37 °C and 150 r/min and then was induced with 1 mM IPTG for 1 h prior to beingharvested. The cells were harvested by centrifugation at 8,000 r/min for 15 min, washedtwice with 0.1 M phosphate sodium buffer (pH 7.0), and resuspended in the same buffer toOD600=2.0, corresponding to a concentration of 4.0 g/L cell dry weight.

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Optimization of 4- and 7-Chloroindole Biotransformation by Strain PHIND

All biotransformation reactions were conducted in 75-mL Erlenmeyer flasks with a 20-mLworking volume at 30 °C and 150 r/min for 3 h. The reaction mixtures consisted of 20 mLwhole cell suspensions with 1 mM glucose and the desired concentration of 4- and 7-chloroindole using dimethyl formamide as a cosolvent. All of the experiments wereperformed in three duplicates, and the average values were used in calculations.

The optimal conditions for biotransformation were determined by RSM. Four importantparameters, strain PHIND inoculums (factor A), 4- or 7-chloroindole concentration (factor B),pH (factor C), and glucose concentration (factor D), were chosen as the independent vari-ables, and the biotransformation rate was set as the dependent response variable (Y). Each ofthe independent variables was studied at five different levels with a total of 30 experiments(Table 1).

Biotransformation Kinetic Assays for Strain PHIND

The reactions were conducted under the optimal conditions obtained above except thevarying concentrations of 4- and 7-chloroindole (100 to 400 μM). The reactions werequenched by the addition of 1 mL of 0.5 M aqueous NaOH after 30 min. Theformation of colored products was measured by HPLC. Kinetic data were calculatedfrom the velocities with the Michaelis–Menten equation. One unit (U) of the whole cellreaction rate was defined as the amount of dry cells able to produce 1 μM products permin [15].

Chemical Analysis and Products Identification

For substrates and products analysis, the reaction mixtures were acidified with 0.1 mL HCl(1 M) and centrifuged (8,739×g, 15 min), and the supernatants were extracted twice with anequal volume of ethyl acetate. HPLC (Shimadzu Prominence LC-20A, Japan) wasconducted to quantify the residue of the substrates. Filtered samples (10 μL) were injectedinto a Hypersil ODS2 column (5 μm, 250×4.6 mm) with CH3OH/H2O as mobile phase. Alinear gradient of 35–45 % CH3OH (v/v, in H2O) over 20 min was used with a flow rate of1 mL/min, and UV detection was at 276 nm. The products were identified by HPLC-MS(Hewlett Packard 1100 MSD, America). HPLC was done on a RX-C18 column (150×2.1 mm; Agilent, CA, USA) with CH3OH (in H2O) as mobile phase containing 0.1 %formic acid. A linear gradient of 35–45 % CH3OH (v/v, in H2O) over 20 min was used with aflow rate of 0.2 mL/min, and UV detection was at 280 nm. Mass spectra were performed ona Finnigan TSQ 7000 triple quadrupole mass spectrometer equipped with a standard API-ESin negative or positive ion mode.

Results and Discussion

Optimization of 4- and 7-Chloroindole Biotransformation by Strain PHIND

In order to optimize the process of 4- and 7-chloroindole biotransformation, RSM wasused to estimate the optimal conditions, and four factors were selected. The experimen-tal and predicted biotransformation rate of 4- and 7-chloroindole with 30 groups ofindependent experiments was shown in Table 1, respectively. According to the multiple

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regression analysis, the following second-order polynomial equation for 4-chloroindolebiotransformation was described as Eq. (1):

Y1 ¼ �74:46þ 62:88Aþ 0:24Bþ 15:51C þ 52:41D� 0:14ABþ 1:25AC � 1:36ADþ0:11BC � 0:22BD� 2:63CD� 13:54A2 � 0:005B2 � 1:72C2 � 4:61D2

ð1Þ

Table 1 The design with experimental and predicted values of biotransformation rate

No. Factor A Factor B Factor C Factor D Biotransformation rate (%)

4-Chloroindole 7-Chloroindole(OD) (mg/L) (mmol/L)

Experimental Predicted Experimental Predicted

1 2.00 75.00 9.00 1.00 57.67 66.87 24.16 50.13

2 2.50 50.00 5.00 1.50 78.02 84.14 37.95 53.05

3 2.00 125.00 7.00 1.00 42.54 49.78 12.45 14.23

4 1.00 50.00 9.00 1.50 40.41 41.84 5.94 11.15

5 1.00 100.00 9.00 0.50 43.57 45.61 9.52 5.46

6 2.50 50.00 9.00 1.50 68.22 68.34 44.50 44.61

7 2.50 100.00 5.00 1.50 53.57 51.64 20.26 31.55

8 1.00 100.00 9.00 1.50 53.57 41.84 10.88 2.10

9 2.50 100.00 9.00 1.50 44.87 57.84 25.61 24.31

10 1.00 50.00 5.00 1.50 64.11 65.11 20.51 22.65

11 2.50 50.00 9.00 0.50 62.00 63.15 25.61 28.21

12 2.50 100.00 5.00 0.50 43.95 46.94 18.67 20.93

13 0.00 75.00 7.00 1.00 0.00 0.00 0.00 0.00

14 2.00 75.00 7.00 1.00 75.82 74.78 79.48 76.63

15 1.00 100.00 5.00 0.50 39.63 36.38 9.91 12.04

16 2.00 75.00 7.00 1.00 74.57 74.78 79.04 76.63

17 2.00 75.00 3.00 1.00 50.58 49.24 3.31 −8.3718 2.50 100.00 9.00 0.50 70.57 63.65 21.03 17.41

19 1.00 100.00 5.00 1.50 38.82 43.11 10.81 12.40

20 2.00 75.00 7.00 1.00 76.16 74.78 79.00 76.63

21 2.00 75.00 7.00 2.00 79.80 75.81 57.86 47.42

22 2.00 75.00 7.00 1.00 75.86 74.78 80.95 76.63

23 2.00 75.00 7.00 0.00 59.57 64.56 21.09 27.24

24 2.00 75.00 7.00 1.00 75.90 74.78 76.33 76.63

25 1.00 50.00 9.00 0.50 36.78 34.61 10.71 5.01

26 2.00 75.00 7.00 1.00 75.46 74.78 79.55 76.63

27 1.00 50.00 5.00 0.50 55.00 47.38 16.53 12.79

28 2.50 50.00 5.00 0.50 60.83 68.45 20.58 32.93

29 2.00 25.00 7.00 1.00 79.01 74.78 47.67 39.03

30 3.50 75.00 7.00 1.00 60.42 52.70 67.77 57.37

Factor A indicates strain PHIND inoculums; factor B, 4- and 7-chloroindole concentration; factor C, pH; andfactor D, glucose concentration

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ANOVA analysis was used to check the quality of the model. The F statistic and P valueof the model were found to be 10.19 and <0.0001, respectively, indicating that the modelwas adequate enough. Moreover, the regression analysis of the data showed that thecoefficient of determination R2 value was about 0.9049, which implied that more than90 % variability in response could be explained by this model (Table 2). The adjusteddetermination coefficient R2 was 0.8160, which also illuminated the significance of themodel. For biotransformation conditions, the P value of A, B, BC, A2, and C2 was significantfactors for their P value less than 0.05. However, P value of C, D, AB, AC, AD, BD, CD, andD2 was larger than 0.05, which suggested that this was not significant factors (Table 3).

As shown in Fig. 1a, b, the biotransformation rate of 4-chloroindole was improved, whenstrain PHIND inoculums (factor A) were increased from 1.00 to 2.50 (OD600). However, withstrain PHIND inoculums increased further (2.50 to 3.00), the biotransformation rate of 4-chloroindole decreased obviously. It was suggested that strain PHIND inoculums were one ofthe most important factors for its P value (0.0299) less than 0.05 (Table 3), which wasconsistent with the previous report [16].

From Table 3, 4-chloroindole concentration (factor B) was another necessary parameterwith the P value of 0.0007. Based on Fig. 1b, with the increase of 4-chloroindole concen-tration, biotransformation rate of 4-chloroindole decreased, which might be due to thetoxicity of 4-chloroindole to the strain PHIND. Zhang et al. also found that all the halogen-substituted indoles showed some cytotoxicity towards bacteria [13].

It was suggested that pH was not a significant parameter (P value=0.5951) (Table 3).When pH was from 5.00 to 7.39, the biotransformation rate of 4-chloroindole was slightly

Table 2 ANOVA analysis for two models

Model SS Mean square F value P value R2

Model 1 8,364.04 597.43 9.88 <0.0001 0.9022

Model 2 20,273.71 1,448.12 10.85 <0.0001 0.9101

Table 3 ANOVA analysis fordifferent factors Factor P value

4-Chloroindole 7-Chloroindole

A 0.0299 0.0679

B 0.0007 0.0225

C 0.5951 0.2112

D 0.1086 0.0585

AB 0.1893 0.3144

AC 0.3478 0.7927

AD 0.7934 0.3803

BC 0.0127 0.9139

BD 0.1776 0.4216

CD 0.1970 0.7512

A2 <0.0001 0.0005

B2 0.0433 <0.0001

C2 0.0030 <0.0001

D2 0.4499 0.0005

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increased, which was different from that at higher pH values (Fig. 1a). Strain PHIND

exhibited high biotransformation rate at pH 7.39. It was indicated that strain PHIND couldtolerate wider pH ranges (5.00–9.00) with high efficiency. Similar result was obtained in thebiosynthesis of indigo by recombinant E. coli harboring fmo gene [17].

The effects of glucose concentration on 4-chloroindole biotransformation were shownin Fig. 1c. When the glucose concentration was increased from 0.5 to 1.26 mmol/L, thebiotransformation rate of 4-chloroindole was improved, whereas with further increasedglucose concentration, the biotransformation rate was kept stable. As previouslyreported, glucose could promote the biotransformation/degradation because it couldinduce the reduced nucleotides (NADH, FADH), which were the redox mediatorsinvolved in the catalysis/degradation of aromatic compounds [18–20]. It might bepresumed that the addition of glucose could provide NADH as a cofactor for phenolhydroxylase.

The quadratic model 1 predicted that the maximal biotransformation rate of 4-chloroindole was 79.04 % in 3 h; when the PHIND inoculums (factor A) was 2.23(OD600), 4-chloroindole concentration (factor B) was 57.42 mg/L, pH (factor C) was 7.39,and glucose concentration (factor D) was 1.26 mmol/L. Validation experiment wasperformed in triplicate tests to verify the predicted results. Under the optimal biotransfor-mation conditions, the biotransformation rate was 78.66 %, which was a good agreementwith the predicted value.

The second-order polynomial Eq. (2) was used to estimate the biotransformation of 7-chloroindole under different conditions.

Y2 ¼ �424:13þ 62:14Aþ 3:20Bþ 78:21C þ 95:77D� 0:15ABþ 0:51AC þ 0:684ADþ0:006BC � 0:19BD� 0:93CD� 13:48A2 � 0:2B2 � 5:76C2 � 39:30D2

ð2ÞFor 7-chloroindole biotransformation, the P value of model 2, factors A, B, A2, C2, and D2

were less than 0.05 (Tables 2 and 3), which suggested that they were significant factors. Thecoefficient of determination R2 and adjusted R2 were found to be 0.9333 and 0.8710,respectively.

The response surface graphs of 7-chloroindole biotransformation were shown in Fig. 2a–c,and the results obtained were similar with that of 4-chloroindole biotransformation. The optimalconditions of biotransformation were obtained by the regression analysis of Eq. (2) as in-oculums of strain PHIND 2.34 (OD600), 7-chloroindole concentration 66.51 mg/L, pH 6.84, andglucose concentration 1.18 mmol/L. Under the optimal biotransformation conditions, thebiotransformation rate was 78.89 % within 3 h, which was consistent with the model predictionof about 77.56 %.

Biotransformation Kinetic Assays for Strain PHIND

To provide insight into the catalytic activity for strain PHIND, the biotransformation kineticassays with 4- and 7-chloroindole were determined by the whole cells of strain PHIND aspreviously described [15]. Strain PHIND exhibited the high formation rate for 7,7′-dichloroindigo (1.35 U/mg cell dry weight), which was 1.14-fold higher than that for 4,4′-dichloroindigo (1.18 U/mg cell dry weight). The Km values of strain PHIND with 4- and 7-chloroindole were calculated as 149.72 μM for 4-chloroindole and 163.04 μM for 7-chloroindole. It was suggested that strain PHIND showed the higher affinity with 4-chloroindole than that with 7-chloroindole.

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(a)

(b)

(c)

Fig. 1 Response surface plot of4-chloroindole biotransformation.a The effects of strain PHIND

inoculums and 4-chloroindoleconcentration, b the effects ofstrain PHIND inoculums and pH,c the effects of strain PHIND

inoculums and glucoseconcentration

Appl Biochem Biotechnol (2013) 170:951–961 957

(a)

(b)

(c)

Fig. 2 Response surface plot of7-chloroindole biotransformation.a The effects of strain PHIND

inoculums and pH, b the effectsof 7-chloroindole concentrationand pH, c the effects of pH andglucose concentration

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Mechanism of 7-Chloroindole Biotransformation

The general pathway for indole biotransformation was that the pyrrole ring (positions 2 and3) was oxidized, and one of the positions was connected to another monomer [21]. 7-Chloroindole was a π-excessive, planar, heteroaromatic compound, and the reactivity of itsC-3 position towards various electrophiles was owing to the significant delocalization ofelectron density from nitrogen to carbon atoms in the pyrrole ring [21]. The products formedfrom 7-chloroindole biotransformation were analyzed by HPLC-MS. The HPLC retentiontime (tr) of product I was 25 min, and its mass spectrum was primarily characterized by amolecular ion at m/z 166.1 ([M−]), which should be identified as 7-chloro-indoxyl (Fig. 3).The molecular ion and retention time (tr) of product II was m/z 331.1 ([M−]) and 39 min,which was designated as 7,7′-dichloroindigo with LC-MS and 1H NMR analysis as de-scribed previously [14]. The color products derived from 7-chloroindole by differentoxygenases have also been investigated. Oxidation of 7-chloroindole by naphthalenedioxygenase and toluene dioxygenase resulted in the formation of two color products (blueand purple) [12]. At least two color products have been produced from 7-chloroindole

m/z100 200 300 4000

20

40

60

80

100

*MSD1 SPC, time=25.952:27.021 of 11042803.D API-ES, Neg, Scan, 50

Max: 3266 166

.1

333

.0 3

35.0

226

.1

168

.0

202

.0

227

.9

334

.1

167

.1

151

.1

264

.0

248

.0

112

.9

62.

0

280

.0

200

.9

Fig. 3 Mass spectra of products formed from 7-chloroindole biotransformation by strain PHIND

HN

Cl

C-3 oxidation

HN

Cl

HN

Cl

OH

O

DimenzationHN

Cl

O

NH

Cl

O

I II

O2 H2ONADH NAD+

O2 H2O

Fig. 4 The proposed pathways for the biotransformation of 7-chloroindole by strain PHIND. I 7-Chloro-indoxyl, II 7,7′-dichloroindigo

Appl Biochem Biotechnol (2013) 170:951–961 959

catalyzed by phenol hydroxylases from Pseudomonas sp. KL28 and KL33 [13]. In contrastto other oxygenases, only one color product, 7,7′-dichloroindigo, was formed by strainPHIND, which showed that strain PHIND possessed higher region specificity for 7-chloroindole. Therefore, according to the HPLC-MS and 1H NMR analysis, the proposedpathway for the biotransformation of 7-chloroindole by strain PHIND was shown in Fig. 4 aspreviously reported [14]. Firstly, 7-chloroindole was hydroxylated at C-3 position to form 7-chloro-indoxyl (product I). Then, two molecules of 7-chloro-indoxyl were polymerized at C-2 position to form 7,7′-dichloroindigo (product II, 00A0).

Conclusions

The whole cells of strain PHIND were used to biosynthesize chloro-substituted indigoidsfrom indoles for the first time. Response surface methodology was adopted to estimate theoptimal biotransformation conditions. Kinetic assays indicated that strain PHIND showedhigh catalytic efficiency for 4- and 7-chloroindole. It was also proved that strain PHIND

possessed higher region specificity for 7-chloroindole. Our work should show high insightsinto the potential of chloro-substituted indigoids production by phenol hydroxylase fromgenus Arthrobacter, which will pave way to novel avenues in green chemistry.

Acknowledgments This work was supported by the National Creative Research Group from the NationalNatural Science Foundation of China (no. 51121062), the National Natural Science Foundation of China (nos.51108120, 51078054, and 51178139), and the 4th Special Financial Grant from the China PostdoctoralScience Foundation (no. 201104430).

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