Facile and Efficient Acylation of Bioflavonoids Using Whole-Cell

Research Article pubs.acs.org/journal/ascecg

Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX-XXX

Facile and Efficient Acylation of Bioflavonoids Using Whole-Cell Biocatalysts in Organic Solvents Xuan Xin,† Xiao-Feng Li,*,† Xinglong Xiao,† Yuqian Tang,† and Guanglei Zhao*,§ †

College of Food Science and Engineering, South China University of Technology, Wushan Road 381, Guangzhou, Guangdong 510640, China § State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Wushan Road 381, Guangzhou, Guangdong 510640, China S Supporting Information *

ABSTRACT: Bioflavonoids have many biological and pharmacological activities, but many of them display low bioavailability after oral administration owing to poor lipophilicity of glycoside forms. To improve their lipophilicity, as well as their bioactivity, a new efficient approach was developed for fatty acylmodification of four vitamin P-like bioflavonoids (troxerutin, rutin, hesperidin, and naringin) using whole-cell biocatalysts in binary solvent systems. Using troxerutin as a typical important bioflavonoid, the acylation activities of 15 strains of different sources were evaluated and Pseudomonas aeruginosa GIM 1.46 showed the highest acylation activity in nonaqueous mediums. 13 C NMR, ESI-MS, and FT-IR analysis confirmed that P. aeruginosa catalyzed acylation of troxerutin to produce troxerutin monopropionate (dominant product) and troxerutin dipropionate. In a binary system of n-heptane and pyridine, the cells showed much higher catalytic activity and operational stability than in other solvents, attributing to a proper increase in permeability of cell envelopes and cell viability by using the binary organic solvents. The optimal n-heptane concentration, molar ratio of vinyl propionate to troxerutin, catalyst dosage, and reaction temperature were found as 35%, 30:1, 50 mg/mL, and 40 °C, respectively, under which the troxerutin conversion and the monoester yield reached 94% and 81.1%, respectively. KEYWORDS: Troxerutin, Biocatalysis, Acylation, Organic solvent, Pseudomonas aeruginosa

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INTRODUCTION Bioflavonoids, commonly known as Vitamin P-like compounds, encompass a diverse set of natural compounds such as rutin, hesperidin and their derivatives. These compounds have aroused considerable attention in nutraceutical, pharmaceutical, and cosmetics industries because of their health-promoting properties, including antioxidative, anti-inflammatory, and blood vessel permeability-reducing activities.1 Unfortunately, the applications of these bioflavonoid glycosides were vastly restricted because of the low lipophilicity, causing poor absorption after oral administration.2 For example, troxerutin, 3′,4′,7-tris[O-(2-hydroxyethyl)] rutin, possesses multiple bioactivities and shows countless application potentials.3−5 But its poor lipophilicity results in the poor absorption through the gastrointestinal tract.6 To date, lots of effort have been done on increasing their lipophilicity and two kinds of strategies were proposed either by reconstruction of dosage form to develop a W/O microemulsion, or by acyl modification to produce flavonoid esters via chemical/enzymatic catalysis.7−9 Since the reconstruction of stable dosage forms needs massive surfactants, the chemical or enzymatic synthesis of flavonoid esters has gained much more attentions. Compared to the chemical methods, enzymatic methods are more eco-friendly © XXXX American Chemical Society

and have higher selectivity under mild reaction conditions. However, the enzymatic processes are not yet industrially feasible due to the high cost of pure enzymes and the limited number of commercially available lipases. Whole-cell catalyzed synthesis of flavonoid esters is a potentially attractive replacement for an enzymatic process, as the cell biocatalysts have more advantages than the isolated enzymes, such as recycling of expensive cofactors, catalyzing multistep cascade reactions, and directly using cell-bound enzymes without separation, purification or immobilization of enzymes.10,11 In addition, cell membrane offers a natural protective barrier for enzymes, which impedes conformational changes of enzyme proteins and thus avoids the loss of enzyme activity in nonaqueous solvents.12 One of the hurdles, however, in microbial whole-cell based processes may include the difficulty in screening whole-cells with high catalytic efficiency from numerous strains/species of microorganisms, because different microorganism possesses different substrate specificities.13 In addition, organic solvents are still widely used in Received: August 1, 2017 Revised: September 16, 2017 Published: October 11, 2017 A

DOI: 10.1021/acssuschemeng.7b02628 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Scheme 1. Transesterification of Troxerutin with Vinyl Propionate Catalyzed by Whole-Cell Catalyst: (A) Troxerutin Structure with Numbered Carbon Atoms and (B) Acylation Process of Troxerutin

China). Troxerutin, rutin, hesperidin and naringin were of 97% purity and purchased from Aladdin (Los Angeles, Southern California, USA). Vinyl propionate (VP, 98% purity) was purchased from TCI (Tokyo, Japan). All other chemicals were obtained from commercial sources and were of the highest available purity. Cell Culture Conditions and Preparation of Whole-Cell Biocatalyst. Bacterial cells were activated in a nutrient broth medium at 37 °C and 180 rpm for 24 h. Mycotic cells were activated in potato dextrose agar (PDA) culture medium at 28 °C for 60 h. And then the seed culture (2%, v/v) was inoculated in the culture medium containing 5.0 g/L yeast extract, 5.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, and 5.0 g/L soybean oil. The cultivation was carried out in 500 mL flasks with 200 mL culture medium on a rotary shaker at 37 °C and 180 rpm for 48 h. To obtain the whole-cell biocatalysts, the bacterial cells were harvested by centrifugation, and then the supernatant was removed. And, the mycotic cells were harvested by vacuum filtration. Then the pellet was washed twice with distilled water to remove the residual medium from the cell surfaces. The washed pellet was freeze-dried in vacuum, ground into powder, and then stored at 4 °C. General Procedure for Whole-Cell-Mediated Acylation of Bioflavonoids. In a typical experiment, 1 mL of organic solvents containing 30 mM troxerutin, 600 mM VP, and 40 mg/mL whole-cell catalyst was added into a 5 mL Erlenmeyer flask (rubber serum cap) and incubated by shaking 180 rpm at 40 °C for 120 h. Aliquots (20 μL) were withdrawn at specified time intervals from the reaction mixture, diluted 50 times with methanol, and then analyzed with HPLC. Each reaction was conducted in triplicate. Meanwhile, the control experiments were carried out in the absence of the cell biocatalyst with different organic solvents. To structure identification of product, the reaction mixture with scaling up was centrifuged to remove cell pellet. The reaction liquid was purified with methanol via reduced pressure distillation and then was isolated to different component with thin-layer chromatography (TLC) method. After crystallization under vacuum drying, two products were obtained as brown powders. Membrane Permeability and SEM Analysis of the Cells Treated with Different Organic Solvents. In a typical experiment, different organic solvent or Tris-HCl buffer (20 mM, pH 7.2) containing Pseudomonas aeruginosa GIM 1.46 cells (50 mg/mL) were incubated in a Erlenmeyer flask (rubber serum cap) at 180 rpm and 30

biocatalytic processes, but they would alter the membrane integrity and morphology of the cells.14 Especially, for polar substrate-involved reactions, there is a contradiction between “the use of polar organic solvents to well dissolve the hydrophilic substrates” and “toxicity of polar organic solvents to microbial cells”.15 Hence, the choice of organic solvents is also a key issue for nonaqueous biocatalysis, which affects not only the substrate solubility, but also the catalytic activity of whole-cells. In the present work, we developed, for the first time, a new facile and efficient approach for selective acylation of vitamin Plike compounds, including troxerutin, rutin, hesperidin and naringin, using whole-cells as biocatalysts. To the best of our knowledge, no researches have been reported concerning the whole-cell catalyzed acylation of those compounds. To approach the above-mentioned “solvent-cell activity” problem, we developed a binary solvent system containing both a hydrophobic and hydrophilic solvent. To a better understand of the bioprocess, effects of solvents and key reaction factors on the catalytic performance and morphologies of the cells were initially evaluated using troxerutin, a multipotent bioflavonoid, as a model substrate (Scheme 1). This strategy flexibly adjusts the hydrophobicity of the reaction and cell permeability by altering the solvents to achieve selective ester synthesis. The recycling of both the cells and solvents enables the whole process more sustainable and cost-effective than the previously reported chemical or enzymatic synthesis.

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EXPERIMENTAL SECTION

Microorganisms and Chemicals. Pseudomonas stutzeri GIM 1.273, Pseudomonas cepacia GIM 1.139, Pseudomonas putida GIM 1.193, Pseudomonas f luorescens GIM 1.209, Pseudomonas aeruginosa GIM 1.46, Bacillus subtilis GIM 1.135, Bacillus megatherium GIM 1.13, Rhizopus oligosporus GIM 3.515, Aspergillus niger GIM 3.25, Penicillium citrinum GIM 3.100, Rhizopus chinensis GIM 3.440, Rhizomucor miehei GIM 3.544, Rhizopus oryzae GIM 3.509, Aspergillus oryzae GIM 3.5232, Geotrichum candidum GIM 2.21 were purchased from GDIM (Guangdong institute of Microbiology, Guangzhou, Guangdong, B


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ACS Sustainable Chemistry & Engineering Table 1. Acylation of Troxerutin with VP Catalyzed by Different Strainsa strains Pseudomonas stutzeri GIM1.273 Pseudomonas cepacia GIM1.139 Pseudomonas putida GIM1.193 Pseudomonas f luorescens GIM1.209 Pseudomonas aeruginosa GIM1.46 Bacillus subtilis GIM1.135 Bacillus megatherium GIM1.13 Rhizopus oligosporus GIM3.515 Aspergillus niger GIM3.25 Penicillium citrinum GIM3.100 Rhizopus chinensis GIM3.440 Rhizomucor miehei GIM3.544 Rhizopus oryzae GIM3.509 Aspergillus oryzae GIM3.5232 Geotrichum candidum GIM2.21 a

biomass (g/L)

V0 (mmol/L·h)

conversion (%)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.01 ± 0.01

58.2 ± 0.06

0.34 0.79 0.52 0.33 0.62 0.80 0.46 4.59 3.46 0.92 3.80 2.11 5.30 5.52 6.23

0.05 0.13 0.27 0.04 0.08 0.09 0.12 0.32 0.32 0.12 0.52 0.22 0.04 0.18 0.13

NAb NA NA

30.9 ± 0.56

NA NA

NA NA 5.8 ± 0.01 62.1 ± 0.12 8.3 ± 0.05

2.18 ± 0.03 NA NA

100 80.2 ± 0.28 82.5 ± 0.70

NA 0.59 1.06 0.22 1.23 1.12 0.23 1.87 1.40

± ± ± ± ± ± ± ±

monoester regioselectivity (%)

0.01 0.02 0.01 0.04 0.02 0.01 0.06 0.02

NA 17.9 22.9 12.4 24.5 23.5 15.8 35.2 27.9

± ± ± ± ± ± ± ±

0.15 0.13 0.07 0.11 0.11 0.08 0.23 0.16

82.0 83.8 82.1 81.5 81.2 82.6 81.4 82.6

± ± ± ± ± ± ± ±

0.99 1.13 0.14 0.71 0.28 0.85 0.56 0.95

The experiments were performed in triplicate, and the data are presented as the mean ± SD. bNA = no activity.

°C. The supernatants were withdrawn from reaction mixture at reacting 48 h and were determined at 260 and 280 nm using ultraviolet spectrophotometer. The net OD260 and OD280 values were calculated by measurement of the 24-h values subtracting the 0-h absorbance values. The release of intracellular matter (nucleic acids and proteins) in reaction media can be monitored by the OD260 and OD280 values to evaluate the effect of organic solvents on cell membrane permeability. The cell pellet was also freeze-dried in vacuum and then gold-plated at cell surfaces. The morphology of cells was analyzed using a ZEISS EVO 18 scanning electron microscope (ZEISS Corp., Germany). The magnification was 5 K in SEM micrographs. Cell Viability Assay. The cell viability was measured by determining the glucose metabolic activity retention (MAR) of cells after being exposed to different organic solvents for a certain time. The details of MAR measurement are provided in the Supporting Information. Determination of Polarity Parameter ET(30). The ET(30) value of pure and binary organic solvents was determined according to the method by Li et al.16 Five milligrams of Reichardt’s dye was added into 100 mL of pure or binary solvents. And then the maximal absorption wavelength of this mixture was recorded by a UNIICO WFZUV-2102 PC spectrophotometer (UNICO instruments Co. Ltd., China). The polarity of reaction medium was estimated using the empirical parameter ET(30), which was calculated from the eq 1

⎛c⎞ E T(30) (kcal/mol) = hvN = h⎜ ⎟N ⎝λ⎠

water (62/38, v/v) with glacial acetic acid (0.1%, v/v) was used as the mobile phase with a flow rate of 0.9 mL/min. The retention time for troxerutin, troxerutin monopropionate (TME) and troxerutin dipropionate (TDE) were 3.6, 5.2, and 7.3 min, respectively. The substrate solubility was determined by HPLC analysis using its saturated solutions in different solvents at 25 °C. The conversion of troxerutin and the yield of TME or TDE were calculated using the eqs 2 and 3 given below, respectively. The initial rate and regioselectivity were calculated using the eqs 4 and 5 given below, respectively. The average error for this assay is less than 1.0%.

conversion (%) =

yield (%) =

(A t − A 0) × 100% A0

Pt × 100% S0

V0 (mmol/L· h) =

(3)

(A t − A 0) × 100% t

regioselectivity (%) =

A pi A total

(2)

× 100%

(4)

(5)

where At = the peak area of troxerutin after reaction, A0 = the peak area of troxerutin before reaction, Pt = the molarity of TME or TDE after reaction (mM), S0 = the initial molarity of troxerutin (mM), t = the reaction time (h), APi = the peak area of target product (pi), and Atotal = the sum of peak area of all products. Structural Characterization of the Products. The acylation position of the products were determined by 13C NMR (Bruker AVANCE Digital 400 MHz Nuclear Magnetic Resonance Spectrometer, Bruker Co., Germany) at 100 MHz. DMSO-d6 was used to dissolve substrate and product and chemical shifts were indicated in ppm shift. The mass spectra of product were obtained on an Agilent 1290/BrukerMaXis Impact Plus ESI Mass Spectrometer with a spray voltage of 4.5 kV (Bruker Co., Germany). The characteristic peak of CO was determined by VERTEX 33 Fourier Transform Infrared Spectrometer (Bruker Co., Germany). Troxerutin. 13C NMR (DMSO-d6, 100 MHz) δ: 177.93 (C-4), 165.11 (C-7), 161.29 (C-9), 157.01 (C-5), 156.89 (C-2), 151.37 (C4′), 148.00 (C-3′), 134.16 (C-3), 123.03 (C-1′), 122.87 (C-6′), 114.99 (C-5′), 113.32 (C-2′), 105.52 (C-10), 101.79 (C-1″), 101.37 (C-1‴), 98.83 (C-6), 93.34 (C-8), 76.87 (C-3′′), 76.42 (C-5′′), 74.64 (C-2′′), 72.24 (C-4′′′), 71.08 (C-3′′′), 71.08 (C-2′′′), 70.93 (C-4′′), 70.83 (CA′), 70.79 (C-A′), 70.67 (C-A), 68.73 (C-5′′′), 67.61 (C-6′′), 60.05 (C−B′), 60.00 (C−B′), 59.79 (C−B), 18.14 (C-6′′′). FT-IR (KBr, cm−1): 1723.34. ESI-MS (m/z): 765.2212 (M + Na)+.

(1)

where h = the Planck’s constant, v = the light frequency, c = the velocity of light, λ = the maximal absorption wavelength, and N = Avogadro’s number. Operational Stability of the Cells. Two milliliters of 35% nheptane-pyridine system containing 30 mM troxerutin, 900 mM VP, and 50 mg/mL whole-cell catalyst was added into a 10 mL Erlenmeyer flask (rubber serum cap). Then the reaction was performed in a shaker at 180 rpm and 40 °C and repeated for ten batches (24 h per batch). For each batch, the whole-cell biocatalyst was separated from reaction system and washed three times with distilled water, and then freezedried and added into a fresh reaction system for next batch. Aliquots (20 μL) were withdrawn at specified time intervals from the reaction system of per batch, diluted 50 times with methanol, and then analyzed with HPLC. The relative activity of cells in the first batch was defined as 100%. HPLC Analysis. The reaction mixture was analyzed by RP-HPLC on a 4.6 mm Â 250 mm (5 μm) Zorbax SB-C18 column (Agilent Technologies Co. Ltd., Massachusetts, USA) using a Waters 600E pump and a Waters 2996UV/photodiode array detector (Waters Corp., Massachusetts, USA) at 350 nm. A mixture of methanol and C


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ACS Sustainable Chemistry & Engineering Table 2. Effect of Various Solvents on Acylation of Troxerutin Catalyzed by P. aeruginosa GIM1.46a entry

solvents

ET(30) (kcal/mol)

1 2 3 4 5 6 7 8 9 10

DMSO DMF pyridine acetonitrile-pyridine (1:3, v/v) t-amyl alcohol-pyridine (1:3, v/v) cyclohexane-pyridine (1:3, v/v) n-hexane-pyridine (1:3, v/v) petroleum ether-pyridine (1:3, v/v) n-heptane-pyridine (1:3, v/v) isooctane-pyridine (1:3, v/v)

45.1 43.2 40.5 42.9 40.0 39.5 38.1 38.0 37.3 36.9

solubility (mg/mL)b 783.4 603.9 472.8 416.1 387.7 351.3 345.5 336.2 334.8 320.2

± ± ± ± ± ± ± ± ± ±

2.45 2.10 2.31 0.69 1.02 0.92 0.75 0.69 1.44 1.17

V0 (mmol/L·h) 0 0 0.21 0.22 0.52 1.38 1.83 1.92 2.07 2.00

± ± ± ± ± ± ± ±

0.10 0.05 0.08 0.08 0.09 0.07 0.07 0.11

conversion (%) 0 0 10.2 1.7 18.3 32.5 35.6 37.1 40.1 38.6

± ± ± ± ± ± ± ±

0.13 0.57 0.52 1.59 0.19 0.22 0.34 0.94

monoester regioselectivity (%) 0 0 77.1 74.3 78.5 78.5 77.8 78.1 79.9 78.1

± ± ± ± ± ± ± ±

1.39 1.48 0.28 0.49 0.49 0.07 0.56 0.49

a The reaction time is 48 h. The experiments were performed in triplicate, and the data are presented as the mean ± SD. bThe solubility of troxerutin was determined by HPLC analyses of the saturated solutions at 25 °C.

Troxerutin Monopropionate (TME). 13C NMR (DMSO-d6, 100 MHz) δ: 177.88 (C-4), 165.13 (C-7), 161.43 (C-9), 156.97 (C-5), 156.82 (C-2), 150.85 (C-4′), 148.14 (C-3′), 134.31 (C-3), 123.07 (C1′), 122.88 (C-6′), 115.38 (C-5′), 113.92 (C-2′), 105.6 (C-10), 101.80 (C-1″), 101.35 (C-1‴), 98.92 (C-6), 93.27 (C-8), 76.91 (C-3′′), 76.39 (C-5′′), 74.58 (C-2′′), 72.25 (C-4′′′), 71.18 (C-3′′′), 71.13 (C-2′′′), 70.93 (C-4′′), 67.34 (C-A′), 70.73 (C-A′), 70.64 (C-A), 68.68 (C5′′′), 67.65 (C-6′′), 60.85 (C−B′), 60.00 (C−B′), 59.74 (C−B), 18.13 (C-6′′′), 174.16 (CO), 27.24 (CH3CH2CH2−), 9.41 (CH3−). FTIR (KBr, cm−1): 1730.09 (CO), 2851 (C−H). ESI−MS (m/z): 821.2475 (M + Na)+. Troxerutin Dipropionate (TDE). 13C NMR (DMSO-d6, 100 MHz) δ: 177.82 (C-4), 165.13 (C-7), 161.71 (C-9), 157.01 (C-5), 156.71 (C-2), 150.84 (C-4′), 148.16 (C-3′), 134.41 (C-3), 123.07 (C1′), 123.04 (C-6′), 115.35 (C-5′), 113.90 (C-2′), 105.71 (C-10), 101.83 (C-1″), 101.32 (C-1‴), 98.91 (C-6), 93.12 (C-8), 76.94 (C3′′), 76.41 (C-5′′), 74.61 (C-2′′), 72.29 (C-4′′′), 71.18 (C-3′′′), 71.18 (C-2′′′), 70.88 (C-4′′), 67.33 (C-A′), 70.74 (C-A′), 67.13 (C-A), 68.68 (C-5′′′), 67.63 (C-6′′), 62.85 (C−B′), 60.01 (C−B′), 62.57 (C−B), 18.13 (C-6′′′), 174.07 × 2 (CO), 27.23 × 2 (CH3CH2CH2−), 9.39 (CH3−). FT-IR (KBr, cm−1): 1730.09 (C O), 2851 (C−H). ESI−MS (m/z): 877.2737 (M + Na)+.

P. aeruginosa cells tended to catalyze the formation of TME, while the cell-bound lipases of P. stutzeri cells preferred catalyzing the synthesis of TDE, resulting in different catalytic behaviors of the two kinds of whole-cell biocatalysts. It is worth mentioning that no acylation reaction occurred by some bacterial cells, mainly because the biocatalysts from different origin possessed different substrate recognition characteristics.13,19 The comparison of the biomass and the substrate conversion also illustrated that the relation between growth/propagation of microbes and production of cell-bound lipases was nonsynchronous. Of the 15 strains that were tested, Pseudomonas aeruginosa GIM1.46 showed the highest catalytic capacity, which gave the fastest initial rate (2.18 mmol/L·h) and the highest conversion (62.1%). Thus, Pseudomonas aeruginosa GIM1.46 was selected as the most suitable wholecell biocatalyst for the synthesis of troxerutin propionate. Effects of Organic Solvents on the Catalytic Activity of Pseudomonas aeruginosa GIM1.46 Cells in Acylation of Troxerutin. Organic solvent system is a significant factor for the synthesis of flavonoid ester because it could affect the initial rate and conversion of reaction by changing the activity and stability of enzymes.17,20 Similarly, whole-cell biocatalysis also displayed the solvent dependence and the catalytic activity of cells varied greatly in different organic solvent systems.16 Therefore, the effect of various organic solvent systems on catalytic activity of P. aeruginosa GIM1.46 cells in acylation of troxerutin were investigated (Table 2). In pre-experiments, we found only strongly polar organic solvents can dissolve troxerutin due to its high polarity. However, a strongly polar organic solvent would strip the essential water from active centers of enzyme proteins and thus inactivate them.21,22 As expected (entries 1−3 of Table 2), Pseudomonas aeruginosa GIM1.46 cells only exhibited catalytic activity in pure pyridine, and the initial rate and substrate conversion of the reaction were very low (only 0.21 mmol/L·h and 10.2%, respectively). The use of DMSO and DMF led to complete inhibition of catalytic activities of Pseudomonas aeruginosa GIM1.46 cells. Except for the inhibitory effect of polar organic solvents on enzyme activity, the desolvation of a polar substrate in strong polar solvents is non-negligible.23 It means the polar substrate troxerutin is difficult to leave a highpolar reaction medium and diffuse to the active sites of the enzymes, thus leading to the insufficient substrate molecules surrounding the enzyme proteins. In general, lipases are more stable in water-immiscible solvents than in polar water-miscible solvents.24 Binary solvents containing both water-immiscible solvents and polar water-

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RESULTS AND DISCUSSION Catalytic Behaviors of Different Microbial Whole-Cells in Acylation of Troxerutin. To screen out the whole-cell catalysts that can efficiently catalyze acylation of troxerutin, catalytic behaviors of 15 strains with the capability of producing lipase in the acylation reaction were investigated (Table 1). The results of the control experiments clearly showed that the acylation reactions did not proceed in the absence of the cell biocatalyst in organic solvents. Among seven bacterial strains, the cells of Pseudomonas aeruginosa GIM 1.46 and Pseudomonas stutzeri GIM1.273 showed relatively high catalytic activities, giving two kinds of products (TME and TDE). All molds tested showed certain catalytic activity and the highest troxerutin conversion (35.2%) was obtained by using Aspergillus oryzae GIM 3.5232 cells. The regioselectivity of TME of the fungal cells was in the range of 81−83%. However, interestingly, Pseudomonas stutzeri GIM1.273 preferred to catalyze the reaction to produce TDE with the regioselectivity of 69.1%, while Pseudomonas aeruginosa GIM 1.46 catalyzed the reaction with the dominant product of a TME (regioselectivity of 80.2%). Previous researches confirmed that the geometrical configuration of the activity site of a lipase, the type of organic solvent used and the physicochemical property of the substrates (such as its hydrophobicity, size and location) are the main factors influencing the regioselectivity of a biocatalyst.17−19 Therefore, it could be speculated that the cell-bound lipases of D


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cell membrane permeability and cell morphology were investigated (Table 3 and Figure 1).

miscible solvents may be in favor of maintaining lipase stability as well as the dissolution of polar substrates.25 We then investigated the effects of types and polarities of a serial of binary organic solvents containing pyridine and another waterimmiscible solvent on acylation activity of P. aeruginosa GIM1.46 cells. As illustrated in entry 4 to 10 of Table 2, the whole-cell catalysts showed an obvious solvent dependence in binary solvent systems. Of the seven binary solvents tested, the initial rates and the conversions were higher than those in pure pyridine, except in the acetonitrile-pyridine system. The ET(30) value, a polar parameter of organic solvent, is usually used to describe the polarity of pure or mixed solvent.26 And the ET(30) value accordingly increased when the polarity of tested solvent increased. As shown in Figure S1 of Supporting Information, the initial rate or conversion of the whole-cell catalyzed acylation showed certain correlation with the ET(30) values of the reaction medium. The two reaction indexes were accordingly enhanced when the ET(30) values of the reaction systems were decreased and vice versa. Considering that the substrate solubility is also an important factor for the reaction efficiency, we also tested the changes of substrate solubility in both pure and binary organic solvents. Table 2 showed that the solubility of the substrate was varied with the solvents used and closely related with the polarity of the solvents in terms of their ET(30) values. In the solvents with lower ET(30) values, lower substrate solubility was found. To eliminate the possible influence of solubility limits of the substrate on the reaction, suitable substrate concentrations that were far less than its solubility were adopted in the organic solvents tested, which means that the substrate can be completely dissolved in different solvents and thus the possible limitations caused by substrate solubility was negligible. Consequently, the results in Table 2 should be attributed to that the addition of hydrophobic solvents reduced the polarity of the reaction system, and thus attenuated the inactivation effect of polar solvents on whole-cell lipases. Besides, the addition of hydrophobic solvents benefited the desolvation of the polar substrate troxerutin and promoted the attraction of troxerutin to acyl-enzyme intermediates. It was also found that the faster initial rate (2.07 mmol/L·h) and the higher conversion (40.1%) were obtained in n-heptane-pyridine system rather than in isooctane-pyridine system with a lower polarity. This phenomenon indicated that much higher hydrophobicity of a reaction medium may be unfavorable to the desolvation of the acyl donor, since the acylation of troxerutin belongs to a bisubstrate reaction (troxerutin and vinyl propionate). In addition, the catalytic activity of the cells in different mixed solvents may be associated with the cell membrane-destruction capabilities of different solvents.27 Little influence has been found on regioselectivity of the reaction by the change of mixed solvents. Overall, the n-heptane-pyridine binary solvent was considered as the most suitable reaction medium for the acylation of troxerutin by P. aeruginosa GIM1.46 cells. Effects of Organic Solvents on Membrane Permeability, Surface Morphology, and Viability of the Cells. Organic solvents not only affect the activity and stability of enzymes bounded on/in the cells but also cause loss of the permeability barrier of cell membrane and the changes in cell morphology and structure.14,28 To further understand the influence mechanism of organic solvent on the whole-cell mediated acylation, the effects of different organic solvents on

Table 3. Effect of Various Organic Solvents on the Permeability of Cell Membrane of P. aeruginosa GIM1.46 Cellsa organic solvents

net OD260

net OD280

tris-HCl buffer DMSO DMF pyridine acetonitrile/pyridine (1:3) t-amyl alcohol/pyridine (1:3) cyclohexane/pyridine (1:3) n-hexane/pyridine (1:3) petroleum ether/pyridine (1:3) n-heptane/pyridine (1:3) isooctane/pyridine (1:3)

0.016 1.441 0.502 0.134 0.165 0.112 0.102 0.106 0.104 0.103 0.106

0.024 2.2 1.795 0.176 0.229 0.131 0.129 0.127 0.125 0.121 0.124

a The net OD260 and OD280 values were calculated from 48-h measured values subtracting 0-h absorbance values. The experiments were performed in triplicate, and the data are presented as the mean ± SD.

The OD260 and OD280 value in a reaction medium were used as direct indicators of intracellular components (nucleic acids and proteins) release. As can be seen from Table 3, the OD260 and OD280 values in organic solvent systems were evidently higher than in a Tris-HCl buffer system (as the control), indicating that the organic solvents could increase the cell membrane permeability of Pseudomonas aeruginosa GIM1.46 cells. Appropriate enhancement of cell permeability could reduce the mass transfer resistance. If the integrity of cell membrane was destroyed seriously, however, it would cause the loss of basic function of a cell membrane, the leakage of intracellular materials, the inactivation of cell-bound proteins or even cell lysis.29 The use of DMSO, DMF or acetonitrilepyridine as a reaction medium resulted in higher OD values than in pure pyridine, which means that the destruction of cell membrane was more serious in those polar solvents than that in pyridine. These observations might be closely related to the poor conversion of troxerutin found in reaction media using DMSO, DMF, or acetonitrile-pyridine, since the catalytic activity of whole cells is closely related to the cell membrane integrity.30 The addition of hydrophobic organic solvents could relieve the damage of pure pyridine to cell membrane, in which the OD260 and OD280 values were obviously lower than in those polar solvents. It was speculated that the binary systems containing a hydrophobic organic solvent could act on the cell membrane and result in moderate permeability, thus promoting the substrate/product into and out of the cells and facilitating the biocatalytic reaction. Of all the organic solvents used, the lowest OD260 and OD280 values were obtained in the n-heptanepyridine system, in accordance with the observed best catalytic efficiency of the P. aeruginosa GIM1.46 cells in n-heptanepyridine. Subsequently, the surface morphologies of the cells incubated with different organic solvents were analyzed by SEM Figure 1 illustrated that, after incubating the cells with Tris-HCl buffer (as control) for 48 h, the cells showed the normal shapes of P. aeruginosa GIM1.46 cells with smooth surfaces and full shape (Figure 1A). When the cells were treated by various organic solvents, different damages of cell envelopes, the distortion of cell shape and the wrinkling of cell surfaces could be easily E


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Figure 1. SEM analysis of cell morphology (A−K) and cell viability (L) of P. aeruginosa incubated in different organic solvents (A, Tris-HCl buffer; B, pyridine; C, DMSO; D, DMF; E, acetonitrile-pyridine; F, t-amyl alcohol-pyridine; G, cyclohexane-pyridine; H, n-hexane-pyridine; I, petroleum ether-pyridine; J, n-heptane-pyridine; K, isooctane-pyridine). The experiments were performed in triplicate, and the data are presented as the mean ± SD.

negative bacterium, P. aeruginosa possesses an outer membrane, a thin peptidoglycan layer and a cytoplasmic membrane in its cell envelope, and its cytoplasmic membrane constituted by a

observed in pyridine, DMSO, DMF, and acetonitrile-pyridine systems (Figure 1B−K), indicating a more seriously damage of cells in those media than in other systems. As a kind of GramF


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Figure 2. Effects of volume content of n-heptane (A), molar ration of substrate (B), dosage of whole-cell catalyst (C), and temperature (D) on acylation of troxerutin by Pseudomonas aeruginosa GIM1.46 cells catalysis. (A) Gradual increase of volume content of n-heptane from 10 to 35. (B) Gradual increase of molar ratio of VP to troxerutin from 10 to 40. (C) Gradual increase of whole-cell catalyst dosage from 20 to 80 mg/mL. (D) Gradual increase of temperature from 25 to 55 °C. All the reaction time is 120 h except the reaction time of study on the volume content of nheptane is 24 h. The experiments were performed in triplicate, and the data are presented as the mean ± SD.

phospholipid bilayer forms a matrix for embedding enzymes and transport proteins.31−33 The varied damage degrees of cell envelopes treated by various organic solvents showed that all the tested organic solvents had toxicity effects on the P. aeruginosa cells. Previous studies indicated that the strongly polar organic solvents (such as pyridine, DMSO, and DMF) will closely distribute around the cells, interact with the outer membrane of the cells by intercollision and finally change the cell surface properties.34 When the changes reached a certain degree, the cell membrane would be disrupted, leading to the leakage of cellular contents, as indicated by the OD measurement. Therefore, these observed phenomena (such as depression and wrinkling of cell surface) in Figure 1B−E may be due to that some cellular contents have already leaked out from the cells during the incubation process. Hence, the organic solvents may destroy the integrality of cell to lead to the leakage of effective enzyme molecules, accumulate on the cytoplasmic membrane or inside the cells, and then lead to partial or complete loss of catalytic activities of P. aeruginosa cells in the reaction. The addition of hydrophobic solvents into pyridine decreased the damage of cell envelope and the cell morphologies were similar to the control group (Figure 1F− K), showing a slight wrinkling. This is mainly due to that these binary solvents contain the hydrophobic organic solvents with similar polarity (Table 2) and similar effects on the

permeability of cell membranes (Table 3). The observations in Figure 1 further confirmed that the binary solvents formed by addition of a hydrophobic solvent into pyridine lead to less severe damages of cell envelopes. Besides the toxic effect on cell membrane integrity described above, organic solvents would destroy the basic metabolism of microbial cells due to their natural toxicity. To give further support to the conclusions of the toxic effects of organic solvents on microbial cells, the cell viability in several representative pure or binary solvents were investigated by measuring the glucose metabolic activity retention (MAR) of the cells. As shown in Figure 1L, the organic solvents with high polarity seriously destructed the basic metabolism of cells, resulting in low viability values. The addition of the hydrophobic solvents significantly decreased the toxic effects on the basic metabolism of the cells. The highest viability value was obtained in the most suitable reaction medium (n-heptanepyridine). These results further confirmed that the hydrophobic solvent-containing binary system had a better biocompatibility with the cells than the polar solvents. In addition, the MAR analysis of cells in different solvents was in accordance with the investigation of the catalytic efficiency of the whole-cells (Table 2) and the observation of cell membrane permeability and morphology changes (Table 3 and Figure 1). From the point of view of whole-cell biocatalysis, not only the polarity of solvent G


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Figure 3 showed that the conversion and yield of TME under the optimal reaction conditions were rapidly increased with the

but also the solvent toxicity to cells should be considered in the choice of organic solvents as reaction medium. Effects of Several Crucial Reaction Conditions on Acylation of Troxerutin. From both an economic and environmental viewpoint, there is a continuing need for optimization of a new bioconversion process. Consequently, to further improve the efficiency of the acylation reaction, the effects of several crucial factors, such as volume content of nheptane, molar ration of acyl donor to substrate, dosage of whole-cell catalyst and reaction temperature, were investigated. Previous researches showed that the concentration of hydrophobic solvents markedly affected the acylation activity of a whole-cell catalyst.35 Hence, the effect of volume content of nheptane on whole-cell biocatalysis process was first investigated. As shown in Figure 2A, the contents of hydrophobic solvents clearly affected the activity of whole-cell catalyst. When the volume content of n-heptane was ranged from 10% up to 35%, the initial rate, the conversion of reaction and the yield of TME markedly increased, and the cell viability also increased up to 84.4% (Figure 1L). Further increase in the volume content of n-heptane, however, caused visible insolubility of troxerutin in reaction medium, resulting in a heterogeneous reaction and the low substrate conversions. Figure 2B shows an increase in VP/ Troxerutin molar ration from 10 up to 30 (mol/mol) brought about remarkable rise in the initial rate, the conversion of reaction and the yield of TME. Beyond 30 (mol/mol) the initial rates, conversion and yield were hardly changed. In the acylation reaction, excessive amount of vinyl propionate was considered to be necessary mainly due to the existence of side reactions, the hydrolysis of both the acyl donor vinyl esters and product esters, because the reaction medium was not absolutely anhydrous but contained trace water.16,31 As evident in Figure 2C, the initial rate, the conversion and the yield of TME were obviously increased with the increase of whole-cell catalyst dosage. When the whole-cell catalyst dosage reached 50 mg/ mL, the initial rate, conversion, and the yield of TME was 2.60 mmol/L·h, 94.5%, and 79.9%, respectively. However, further improving the whole-cell catalyst dosage was not available for further immensely increasing the conversion and yield of the reaction. The presence of higher content of whole-cell catalyst may provide more activity sites to form the acyl-enzyme complex, but further increase the dosage of whole-cell catalyst has no significant effect on enhancement of conversion and yield because of the limitation of the substrate amount and the steric hindrance effect.34 It is well-known that reaction temperature affects the catalytic activity and the thermostability of whole-cell biocatalyst, but also influences the reaction equilibrium and the permeability of cell membrane to reduce the mass transfer resistance.35,36 Figure 2D showed the effect of reaction temperature on the P. aeruginosa GIM1.46 cellmediated acylation of troxerutin in the range of 25−55 °C. As indicated, the initial rate, the conversion of reaction and the yield of TME were significantly increased with the increase of reaction temperature from 25 to 40 °C. Exceed the 40 °C, the conversion and the yield of TME rapidly decreased mainly because too high temperature caused the loss of enzymatic activity of whole-cell biocatalyst. From 40 to 50 °C, the initial rate of reaction still has a clear increase from 2.76 mmol/L·h to 3.03 mmol/L·h, this was the relatively high temperature accelerates the collision between the activated substrate and the enzyme in reaction initial stage to lead to the increase of the apparent maximum initial rates.

Figure 3. Time course of conversion of reaction and yield of troxerutin monopropionate. Reaction conditions: 35% n-heptane-pyridine as reaction medium; molar ratio, 30:1; whole-cell catalyst dosage, 50 mg/ mL; temperature, 40 °C. The experiments were performed in triplicate, and the data are presented as the mean ± SD.

increase of reaction time. At reacting 120 h, the conversion and yield of TME reached 94% and 81%, respectively, and then hardly changed by further extending the reaction time. The reaction equilibrium time for catalysis by whole-cell biocatalyst was usually longer than that with free enzymes, because the outer membrane of P. aeruginosa functions as a molecular sieve to prevent molecules with a molecular mass of greater than 600−1000 Da penetrating.31 But when the organic solvent increased the permeability of cell membrane, the reaction rate started to increased. A slight increase in membrane permeability was beneficial to the catalytic efficiency of the cells. It is important to point out that the substrate conversion and ester yield obtained under the optimum conditions reached 94.0% and 81.1%, respectively, which were much higher than the previously reported chemical methods (substrate conversion of 57.0%) or enzymatic methods (ester yield of 59.2%).7,8 Acylation of Vitamin-P Compounds with Different Structures by Pseudomonas aeruginosa GIM1.46 Cells. To explore the catalytic performance of Pseudomonas aeruginosa GIM1.46 cells in substrate recognition, several bioflavonoids possessing similar vitamin-P functions were investigated as substrates. Rutin, hesperidin, and naringin also showed multiple bioactivities including antilipid peroxidation, scavenging free radical, anti-inflammatory, etc.37,38 And their structures also consist of 2-phenyl-chromone (parent nucleus) and disaccharide (aglycon), similar to the structure of troxerutin. As shown in Table 4, rutin and hesperidin were also relatively good substrates for Pseudomonas aeruginosa GIM1.46 cells and only the 4‴-O-propionyl rutin and 4‴-O-propionyl hesperidin were synthesized with a yield 61.5% and 59.2%, respectively. The conversions of rutin and hesperidin (approximately conversion of 60%) were significantly lower than that of troxerutin because of the absence of the primary hydroxyl group. Interestingly, Pseudomonas aeruginosa GIM1.46 cells could catalyze the acylation of naringin more efficiently, producing one product (6″-propionyl naringin) with a higher yield of 87.9%. This may be attributed to that naringin bears only one primary hydroxyl H


Research Article

ACS Sustainable Chemistry & Engineering Table 4. Whole-Cell Biocatalyst-Mediated Acylation of Bioflavonoida

a Reaction condition: 35% n-heptane-pyridine as reaction medium; molar ratio, 30:1; whole-cell catalyst dosage, 50 mg/mL; 40 °C, 180 rpm; reaction time, 120 h. The experiments were performed in triplicate, and the data are presented as the mean ± SD.

the relative activity of P. aeruginosa GIM 1.46 cells rapidly decreased and was below 18% of their original activity at ten batches, because that the integrity of cell membrane would be destroyed seriously by organic solvents and the cell-bound enzymes also might be inactivated by organic solvents during multiple reuses. Despite all this, the results confirmed that the whole-cell biocatalyst possessed superior operational stability to the free enzymes that can only be used for one batch unless an extra successful immobilization. The high operational stability of P. aeruginosa GIM 1.46 cells also indicated that the cost of production can be greatly reduced and made the whole-cell mediated processes more sustainable than those with free enzymes.

group and thus has less steric hindrance when attacking the acyl-enzyme intermediate. It was also found that the regioselectivity of the cells was great improved to above 99% when rutin, hesperidin and naringin were used as substrates. Operational Stability of Pseudomonas aeruginosa GIM1.46 Cells in Acylation of Troxerutin. As can be seen in Figure 4, the relative activity of P. aeruginosa GIM 1.46 cells in n-heptane-pyridine system still retained above 80% after recycling operation for five batches, showing a good operational stability of P. aeruginosa GIM 1.46 cells in the reaction system. This should be ascribed to naturally protective effect of the cellular structure as a natural carrier for the enzymes. And then

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CONCLUSIONS

In this study, we established a new biocatalytic method for facile and efficient synthesis of bioflavonoid esters. This method not only relies on the good catalytic performance of Pseudomonas aeruginosa GIM1.46 but also the proper improvement in both the hydrophobicity of reaction media and the permeability of cell envelope via adjusting the composition of binary organic solvents. With this method, the conversion of troxerutin was up to 94%, which are significantly higher than those with previously reported chemical or enzymatic methods. Microbial cells are natural sustainable catalysts. Moreover, the use the whole-cell biocatalysts avoids the tedious purification and immobilization of pure enzymes. Coupled with high reusability of the cells and recycling capability of organic solvents, this approach can afford dramatic cost reductions and of great interest for sustainable production of bioflavonoidrelated chemicals, and applications in food, pharmaceutical and cosmetics processing. The application of whole-cell catalysis toward other types of flavonoids and their derivatives is being further explored in our laboratory.

Figure 4. Operational stability of Pseudomonas aeruginosa GIM1.46 cells in acylation of troxerutin. Reaction conditions: 35% n-heptanepyridine as reaction medium; molar ratio, 30:1; whole-cell catalyst dosage, 50 mg/mL; temperature, 40 °C; reaction time, 24 h. The experiments were performed in triplicate, and the data are presented as the mean ± SD. I


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02628. Additional experimental section, abbreviations, the relationship of ET(30) value of various organic solvents, and the initial rate of conversion (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: (+86)20-22236819. E-mail: xfl[email protected]. Fax: (+86)20-87112853. *Tel: (+86)20-87111770. E-mail: [email protected]. Fax: (+86)20-87111770. ORCID

Xiao-Feng Li: 0000-0002-0144-6296 Funding

This work was financially supported by the National Natural Science Foundation of China (Nos. 31270636, 21676105), Program for New Century Excellent Talents in University (NCET-12-0192), Self-Determined Research Fund of SCUT from the College Basic Research and Operation of MOE (Nos. 2015ZZ111, 2015ZZ123), and the Science and Technology Program Foundation of Guangdong Province (No. 2015A030401025). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Nos. 31270636, 21676105), Program for New Century Excellent Talents in University (NCET-120192), Self-Determined Research Fund of SCUT from the College Basic Research and Operation of MOE (Nos. 2015ZZ111, 2015ZZ123), and the Science and Technology Program Foundation of Guangdong Province (No. 2015A030401025).

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Facile and Efficient Acylation of Bioflavonoids Using Whole-Cell

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