Biotransformation of Indole to Indigo by the WholeCells of Phenol Hydroxylase Engineered Strainin Biphasic Systems

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

Received: 8 September 2012 /Accepted: 26 December 2012 /Published online: 10 January 2013# Springer Science+Business Media New York 2013

Abstract Biotransformation of indole to indigo in liquid–liquid biphasic systems wasperformed in Escherichia coli cells expressing phenol hydroxylase. It was suggested thatindole could inhibit the cell growth even at low concentration of 0.1 g/L. The critical Log Pfor strain PH_IND was about 5.0. Three different solvents, i.e., decane, dodecane, and dioctylphthalate, were selected as organic phase in biphasic media. The results showed thatdodecane gave the highest yield of indigo (176.4 mg/L), which was more than that of singlephase (90.5 mg/L). The optimal conditions for biotransformation evaluated by responsesurface methodology were as follows: 540.26 mg/L of indole concentration, 42.27 % oforganic phase ratio, and 200 r/min of stirrer speed; under these conditions, the maximalproduction of indigo was 243.51 mg/L. This study proved that the potential application ofstrain PH_IND in the biotransformation of indole to indigo using liquid–liquid biphasicsystems.

Keywords Phenol hydroxylase . Indole . Indigo . Biotransformation . Biphasic systems

Introduction

Indigo is one of the oldest and largest-selling textile dyes for dyeing and printing, which isnot only used as dyes but also serves as the promising kinase inhibitors for its therapeuticvalue [1]. In 1983, Ensley et al. reported the naphthalene dioxygenase expressed in Escher-ichia coli could oxidize indole to indigo and indirubin, which paved a way to the green

Appl Biochem Biotechnol (2013) 169:1088–1097DOI 10.1007/s12010-012-0069-y

S. Shi : F. Ma (*) : T. Sun :A. LiState Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin150090, Chinae-mail: hit_dlut@126.com

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

process for the bio-industrial production of indigo [2]. Since then, several oxygenases, suchas naphthalene dioxygenase, cytochrome P450 monooxygenase, toluene monooxygenase,and phenol hydroxylase (PH) obtained mainly from the genus Pseudomonas sp. have beenstudied to oxidize indole to indigo [2–6]. There have been considerable studies to charac-terize and identify the oxygenases for indigo production [5, 7]. However, the process ofindigo biosynthesis is not economically feasible owing to the very low solubility and thehigh toxicity of indole. Therefore, substrate loading needs to be increased, and the toxicity ofindole has to be minimized to make the process feasible [8].

As a sequence, biotransformation in biphasic systems (water-organic solvent) has beendeveloped, which offers the advantage of increasing substrate loading and maintaining lowconcentration of toxic compounds in the aqueous phase [9]. Recently, much work on thebiphasic systems mainly focused on the biosynthesis of 1-naphthol, 3-methylcatechol,phenol, etc. [10, 11]. Up to now, there have been only two reports about oxidizing indoleto indigo in biphasic systems [8, 12]. For example, Doukyu et al. used the PH and its variant(OST 3410) to oxidize indole to indigo in biphasic systems, but the indigo production wasonly 12 and 52 μg/mL, respectively [12]. Moreover, there is limited information on PH fromArthrobacter sp. strain as biocatalyst for indigo biosynthesis [13]. Therefore, it is importantand necessary to study the use of PH from Arthrobacter sp. strain in indigo biosynthesis viabiphasic systems, which should provide fundamental and useful supports for PH fieldapplication.

The aim of this study was to develop a biphasic system to increase the production ofindigo by PH from Arthrobacter sp. W1. Organic solvents were screened and the toxicity ofindole was also investigated. The effects of indole concentration, organic phase ratio andstirrer speed were optimized by response surface methodology (RSM) using central com-posite design (CCD) method.

Materials and Methods

Chemicals

Indole, indigo, decane, dodecane, dioctyl phthalate, kanamycin, and isopropyl-1-thio-β-D-galactopyranoside (IPTG) were purchased from J&K chemical company (Co., Ltd., Beijing,China). All the other reagents and solvents were of analytical grade.

Bacterial Strain and Growth Conditions

E. coli BL21 (DE3) expressing PH was used in this study, which was designated as strainPH_IND. The strain PH_IND were routinely grown in Luria-Bertani (LB) medium supple-mented with 30 μg/mL kanamycin at 37 °C to an optical density at 600 nm (OD600)of ∼0.4. Expression of PH was induced with 1 mM IPTG. After 1 h of induction,cells were centrifuged, washed twice with 0.1 M phosphate sodium buffer (pH 7.0),and re-suspended in the same buffer to form cell suspensions as whole cell suspen-sions (OD600=2.0).

Toxicity Experiment

The toxicity experiment was carried out as previously described with minor modifications[10]. Fresh LB medium was inoculated with an overnight culture of strain PH_IND until the

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early exponential phase, and indole dissolved in 100 μL dimethyl formamide (DMF) wasadded to the growing cells to obtain final concentration of 0.1 to 1.5 g/L. The culture with orwithout 100 μL DMF was used as the positive control experiment. Owing to the lowsolubility of indole in the aqueous phase, DMF was used to suspend indole. The growthof strain PH_IND was determined by ultraviolet–visible (UV/vis) spectrophotometer (JASCOV-560, Japan) at 600 nm (OD600).

Determination of Critical Log P of Strain PH_IND

Ten kinds of organic solvents, i.e., isooctyl alcohol, chloroform, n-hexane, diphenylmethane,di-n-butyl phthalate, decane, dodecane, n-octane, dioctyl phthalate, and n-hexadecane, witha broad range of Log P (1.67–8.70) were selected; 2 mL inoculums of strain PH_IND wasadded into 250-mL shaken flask with 100 mL fresh LB medium and 20 mL different organicsolvent, respectively. The flasks were incubated at 37 °C and 150 r/min for 24 h. A positivecontrol flask which contained no solvent was also performed. The growth of strain PH_INDwas also monitored by UV/vis and the relative metabolic activity was measured to thecontrol.

Distribution Coefficient Measurement

The measurement of distribution coefficient was carried out as previously described [10].Different organic solvent (2 mL) containing 500 mg/L indole was added to 2 mL phosphatesodium buffer (pH 7.0) in 10 mL microcentrifuge tubes. The tubes were stirred at 37 °C and150 r/min for 1 h. The concentration of indole in the two phases was analyzed by HPLC,respectively. A distribution coefficient was calculated by determining the ratio of the indoleconcentration in organic phase to the indole concentration in the aqueous phase.

Optimization of Indole Biotransformation by Whole Cells of Strain PH_IND

All the biotransformation reactions were carried out in 150 mL Erlenmeyer flasks with aworking volume of 50 mL at 30 °C and 150 r/min. For single-phase biotransformation,whole cell suspensions supplemented with 1 mM glucose were incubated with indole at afinal concentration of 200 mg/L. For biphasic biotransformation, whole cell suspensionssupplemented with 1 mM glucose were added into the desired volume of an organic solventwith a certain concentration of indole to obtain a final volume of 50 mL.

In this work, RSM was used to evaluate the optimal conditions for biotransformation inbiphasic systems, including indole concentration (factor A), organic phase ratio (factor B),and stirrer speed (factor C). The ranges and levels of these three factors were shown inTable 1. The optimal conditions were determined by CCD with 20 groups of independentexperiment (Table 2). For statistical calculations, the following equation was used todescribe the relationship between coded values and real ones:

Xi ¼ Ui � U0ð ÞΔU

ð1Þ

where Xi was the coded value of independent variable, Ui was the real value of theindependent variable, U0 was the center point of Ui, and ΔU was the step change in Ui.Each coefficient was analyzed by “ANOVA.” If the P value (probability>F) was less than

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0.05, the model term and the parameters were considered significant and the adequate modelcould be used in the further experiments.

HPLC Analysis

Indigo production was quantified by HPLC (Agilent 1100, USA) with RX-C18 column(150×2.1 mm). The reaction mixtures were centrifuged to separate the aqueous phase, theorganic phase and the pellet cells. The aqueous phase and the pellet cells were extracted withethyl acetate. Organic phase was injected directly. The HPLC was operated as follows:CH3OH (in H2O) mobile phase containing 0.1 % formic acid, 65–75 % CH3OH linear

Table 1 Conditions and the levels of the optimization experiment design

Factors Symbol Unit − levels + levels

Indole concentration A mg/L 500.00 700.00

Organic phase ratio B % 30.00 60.00

Stirrer speed C r/min 100.00 200.00

Table 2 The design of experimental and predicted values of indigo production

No. Factor Aa (mg/L) Factor Bb (%) Factor Cc (r/min) Indigo concentration (mg/L)

Experimental Predicted

1 1 (700) 1 (60) −1 (100) 170 171.28

2 1 (700) −1 (30) 1 (200) 195 200.38

3 −1 (500) 1 (60) 1 (200) 239.7 241.48

4 0 (600) 0 (45) 0 (150) 204.4 210.08

5 0 (600) 1.67 (70) 0 (150) 188.3 192.38

6 0 (600) 0 (45) 0 (150) 210.6 210.08

7 0 (600) −1.67 (20) 0 (150) 182.2 200.63

8 0 (600) 0 (45) −1.7 (65) 151.7 172.56

9 0 (600) 0 (45) 0 (150) 203.5 210.08

10 −1.69 (431) 0 (45) 0 (150) 182.87 194.94

11 1 (700) −1 (30) −1 (100) 175 177.28

12 1.68 (768) 0 (45) 0 (150) 150.9 162.99

13 0 (600) 0 (45) 0 (150) 202 210.08

14 1 (700) 1 (60) 1 (200) 187.8 199.48

15 0 (600) 0 (45) 1.68 (234) 256 257.49

16 0 (600) 0 (45) 0 (150) 206.2 210.08

17 −1 (500) −1 (30) −1 (100) 180.2 174.28

18 0 (600) 0 (45) 0 (150) 203.87 210.08

19 −1 (500) 1 (60) −1 (100) 171.7 165.28

20 −1 (500) −1 (30) 1 (200) 245.9 245.38

a Indole concentrationb Organic phase ratioc Stirrer speed

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gradient over 20 min; flow rate, 0.2 mL/min; column temperature, 25 °C; and UV detection,242 nm. All of the experiments were performed in three duplicates and the average valueswere used in calculations.

Results and Discussion

Toxicity of Indole for Strain PH_IND

Indole and its derivatives form a class of toxic recalcitrant N-heterocyclic compound that areconsidered as toxic pollutants, which can inhibit the activity of microorganisms [14]. In

Fig. 1 Toxicity of indole for strain PH_IND. Control (squares), control+DMF (circles), 0.1 g/L of indole(triangles), 0.2 g/L of indole (inverted triangles), 0.5 g/L of indole (left-pointing triangles), 1.0 g/L of indole(right-pointing triangles), and 1.5 g/L of indole (diamonds)

Fig. 2 Growth of strain PH_IND in the presence of solvents with different Log P

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order to investigate the toxicity of indole for strain PH_IND, the growth of strain PH_IND withvarying concentrations of indole (0.1 to 1.5 g/L) was studied. As seen from Fig. 1, it wasobvious that indole could inhibit the growth of strain PH_IND even at a low concentration of0.1 g/L. Moreover, the inhibition of growth increased with the concentration of indoleincreasing to 0.5 g/L and no growth was observed, when indole was from 1.0 to 1.5 g/L,which was consistent with the report of Doukyu [8]. Therefore, maintaining low concentra-tion of the substrate was critical for keeping the viability and the activity of cells for thereaction [10].

Determination of Critical Log P of Strain PH_IND

In order to provide insights into the sensitivity of strain PH_IND to the presence of organicsolvents, the critical Log P of strain PH_IND was determined by exposing the cells to ten

Table 3 Distribution coefficients of the solvents

Organic

solventStructure Log P

Partitioned into

organic phase (%)

Distribution

coefficient

Decane 5.25 77.5 3.4

Dodecane 6.10 87.5 7.0

Dioctyl phthalate

O

O

O

O

7.50 65.6 2.0

Fig. 3 Indigo production in an aqueous and biphasic phase by strain PH_IND

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kinds of organic solvents with Log P ranging from 1.67 to 8.70. It was clearly demonstratedthat in the presence of solvent with a Log P below 5.0, the relative metabolic activity of strainPH_IND was lower than 70 % (Fig. 2). It was proved that the solvent with Log P lower than 5.0could inhibit the metabolic activity of strain PH_IND, which might accumulate in the cytoplas-mic membrane and impact the integrity of the cell membrane [15]. However, when the Log P ofsolvent was larger than 5.0, the relative metabolic activity of strain PH_IND was above 90 %.Thus, the critical Log P for strain PH_IND was found to be 5.0. It was an indication that themicroorganism might modify the membrane or the surface properties as well as the activationand/or induction of an active transport system eliminated the solvents from the cell membrane,when they were exposed to solvents [16]. Therefore, decane, dodecane, and dioctyl phthalatewere selected as the biocompatible solvents for their Log P larger than 5.0.

Selection of an Appropriate Organic Phase

As well known, it is a critical step to choose the organic solvent for the proper biphasicmedia [17, 18]. Thus, organic solvents (decane, dodecane, and dioctyl phthalate) withdifferent distribution coefficients and structures were firstly selected (Table 3). Thebiotransformation of indole to indigo in biphasic systems was shown in Fig. 3. Asseen from Fig. 3, the production of indigo were 142.5, 176.4, and 89.3 mg/L usingdecane, dodecane, and dioctyl phthalate as the organic phase in the biphasic systems,respectively. Dodecane with the highest distribution coefficient for indole exhibitedthe highest production of indigo, which was 1.2- and 1.5-fold higher than that ofdecane and dioctyl phthalate, respectively. It was indicated that the dynamics of indolepartitioning into organic phase played a major role in maintaining cell activity andimproving indigo production. Moreover, inefficient partitioning of indole would resultin the accumulation of toxic substrate in the aqueous phase thereby the cellularactivity and indigo production were both decreased [10]. The biotransformation ofindole in an aqueous phase was also investigated as the control, and 90.5 mg/L ofindigo was obtained after 12 h (Fig. 3). Compared with the biotransformation in theaqueous phase, the indigo production was improved 2-fold using dodecane as thesecond phase. Thus, dodecane was selected as the appropriate organic phase for thefurther optimization process.

Table 4 ANOVA analysis for the full quadratic

Source df SS Mean square F value P value (Prob>F)

Model 9 14,117.46 1,568.607 18.22288 <0.0001

A 1 1,935.172 1,935.172 22.48135 0.0008

B 1 44.91758 44.91758 0.521818 0.4866

C 1 8,428.258 8,428.258 97.91308 <0.0001

AB 1 6.105329 6.105329 0.070927 0.7954

AC 1 1,149.601 1,149.601 13.35519 0.0044

BC 1 13.60985 13.60985 0.158109 0.6993

A2 1 1,809.517 1,809.517 21.02158 0.0010

B2 1 353.4769 353.4769 4.106425 0.0702

C2 1 48.35312 48.35312 0.56173 0.4708

df degrees of freedom, SS sum of squares

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Fig. 4 Response surface plot of biotransformation: a effects of indole concentration (factor A) and organicphase ratio (factor B) on indigo productivity; b effects of indole concentration (factor A) and stirrer speed(factor C) on indigo production

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Optimization of Indole Biotransformation by the Whole Cells of Strain PH_IND

The biotransformation conditions were optimized using dodecane as an organic solvent. Inthis study, RSM was applied to optimize the biotransformation conditions and identify therelationship between the response functions and process variables. Indigo production (Y)could be explained by the following second order polynomial equation:

Y ¼ �416:92þ 1:56Aþ 1:44Bþ 1:65Cþ 0:0005AB� 0:002ACþ 0:002BC

� 0:001A2 � 0:02B2 þ 0:0007C2 ð2Þ

Several criteria, such as F statistic and P value were used to check the quality ofmodel and each coefficient [19]. According to the ANOVA analysis, F statistic(18.22) and P value (<0.0001) suggested that the model was adequate enough(Table 4) [20]. The determination coefficient R2 (0.9425) indicated a good fitness ofmodel. For biotransformation conditions, A, C, AC, and A2 were significant factorsdue to their P value (<0.05). However, B, AB, BC, B2, and C2 were not significantfor their P value more than 0.05 (Table 4).

Indole concentration (factor A) was one of the most important factors for its Pvalue (<0.0001) less than 0.05 (Table 4). As shown in Fig. 4a, with the increase ofindole concentration, the production of indigo was increased. It might be due tohigher indole concentration in organic phase, which allowed more indole to releaseinto aqueous, thus the indigo production was improved. However, when indoleconcentration was increased from 550 to 700 mg/L, the indigo production wasdecreased. The production of indigo was improved when the organic phase ratio(factor B) was increased from 30.00 to 42.27 %. As previously reported, the higherorganic phase ratio could enhance the biotransformation by allowing better partition-ing of substrate and product, which minimized its toxicity and improved the concen-tration of products [21]. Further increasing organic phase ratio from 42.27 to 60.00 %,the production of indigo was decreased, which was consistent well with the results ofGarikipati [10]. Stirrer speed (factor C) was another necessary parameter with the Pvalue <0.0001 (Table 4). Indigo production was increased when stirrer speed wasincreased from 100 to 200 r/min (Fig. 4b). It was suggested that higher stirrer speedcould allow more dissolved oxygen and indole to participate in the aqueous phase,which was the similar with the previous report [22].

The predicted optimal values of indole concentration, organic phase ratio and stirrerspeed obtained by the regression analysis (Eq. 2) were as follows: 540.26 mg/L ofindole concentration, 42.27 % of organic phase ratio, and 200.00 r/min of stirrerspeed. Validation experiment was performed in the triplicate under the optimalbiotransformation conditions, and the indigo production was 250.39 mg/L, whichwas a good agreement with the predicted values (243.51 mg/L).

Conclusions

The whole cells of strain PH_IND were used to oxidize indole to indigo in biphasic media. Itwas found that indole could inhibit the growth of cells even at 0.1 g/L, and the critical Log Pof strain PH_IND was about 5.0. It was proved that dodecane was the best organic phase forindole transformation. The optimal production of indigo (243.51 mg/L) was obtained by

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RSM in the biphasic media, which was 2.7-fold higher than that in the aqueous media. Thepresent study suggested that strain PH_IND should be an effective organic solvent-tolerantbiocatalyst in the industrial bio-production of indigo.

Acknowledgments The authors gratefully acknowledge the financial supports from the National NaturalScience Foundation of China (No. 51078054, 51108120, and 51178139), the National Creative ResearchGroup from the National Natural Science Foundation of China (No. 51121062), the 4th China PostdoctoralScience special Foundation (No. 201104430), and the 46th China Postdoctoral Science Foundation (No.20090460901).

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