hydrolysis of tannic acid catalyzed by immobilized ... 186 the time course of tannic acid hydrolysis

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    Hydrolysis of Tannic Acid Catalyzed by Immobilized-Stabilized 1

    Derivatives of Tannase from Lactobacillus Plantarum. 2





    †Instituto de Fermentaciones Industriales. CSIC. c/ Juan de la Cierva 3, 28006 Madrid . Spain 7

    ‡Instituto de Catálisis. CSIC. Campus UAM, 28049 Madrid. Spain 8

    § IMDEA-Alimentación. Campus UAM. 28049 Madrid. Spain 9


    Co-corresponding authors: 11

    Gloria Fernández-Lorente ( Tel: 91.585.48.09; Fax: 91.585.47.60; E-mail:gflorente@ifi.csic.es) 12


    Running Head: Hydrolysis of Tannic Acid. 14






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    Abstract 19

    A recombinant tannase from Lactobacillus plantarum, overexpressed in E. coli, was 20

    purified in a single step by metal chelate affinity chromatography on lowly activated 21

    nickel supports. It was possible to obtain 0.9 grams of a pure enzyme by using only 20 22

    mL of chromatographic support. The pure enzyme was immobilized and stabilized by 23

    multipoint covalent immobilization on highly activated glyoxyl agarose. Derivatives 24

    derivatives obtained by multipoint and multisubunit immobilization were 500- and 25

    1000-fold more stable than both the soluble enzyme and the one point immobilized 26

    enzyme in experiments of thermal and cosolvent inactivation, respectively. In 27

    addition to that, up to 70 mg of pure enzyme were immobilized on 1 gram of wet 28

    support. 29

    The hydrolysis of tannic acid was optimized by using the new immobilized tannase 30

    derivative. The optimal reaction conditions were 30% diglyme, at pH 5.0 and 4 oC. 31

    Under these conditions, it was possible to obtain 47.5 mM of gallic acid from a 5 mM 32

    of tannic acid as substrate. The product was pure as proved by HPLC chromatography. 33

    On the other hand the immobilized biocatalyst preserved more than 95% of its initial 34

    activity after 1 month of incubation under the optimal reaction conditions. 35


    Keywords: purification of recombinant enzymes with poly-His tags, enzymatic 37

    production of pure gallic acid. 38


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    Introduction 40

    Enzymes present important advantages in numerous areas of the Food Chemistry such 41

    as synthesis of prebiotics (1), modification of functional ingredients (2), synthesis of 42

    antioxidants (3), elimination of antinutrients, synthesis of bioactive peptides (4), design 43

    of enzymatic biosensors (5), etc. For the majority of the previously mentioned 44

    applications, utilization of immobilized enzymes is technologically advantageous (6, 7, 45

    8). However, food technology must be so far economically viable, thus immobilized 46

    enzyme derivatives should be excellent in terms of activity, robustness and efficiency, 47

    etc. (9, 10). 48

    The hydrolysis of tannic acid to obtain gallic acid and glucose is an important reaction 49

    in food chemistry (11). The substrate, tannic acid, is an abundant plant residue and it 50

    may be used to prepare different food preservatives as pyrogallol and propyl gallate 51

    (12, 13). In pharmaceutical chemistry gallic acid is also an important intermediate for 52

    the synthesis of the antibacterial drug trimethroprim (14). Practical implementation of 53

    this process requires the preparation of very active and stable immobilized derivatives 54

    of pure tannases able to hydrolyze different gallic-gallic and gallic-glucose bonds 55

    without suffering product inhibition (15-17). 56

    Herein we will describe the preparation of a very active and stable derivative of a 57

    tannase from Lactobacillus plantarum (18). The enzyme was firstly purified to 58

    homogeneity by selective adsorption on small volumes of chelate activated agarose 59

    (19). The subsequent immobilization of the pure protein by covalent multipoint and 60

    multisubunit attachment on glyoxyl agarose stabilized the enzyme (20,21). Finally, the 61

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    optimal conditions for the production of pure and concentrated gallic acid were also 62

    investigated by using the best immobilized derivatives of tannase. 63

















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    Materials and methods 80

    Materials and bacterial strains 81

    Crosslinked 6% agarose beads and cyanogen bromide activated Sepharose 4B (CNBr-82

    agarose) were obtained from GE Healthcare (Uppsala, Sweden). Iminodiacetic acid 83

    disodium salt monohydrate (IDA) and nickel (II) sulphate 6-hydrate and methyl gallate 84

    were purchased from Fluka (Buchs, Switzerland). Tannic acid was obtained from Sigma 85

    (St. Louis, MO, USA). Epichlorohydrin and imidazole were purchased from Merck 86

    (Darmstadt, Germany). All other reagents were of analytical grade. L. plantarum CECT 87

    748T strain was purchased from the Spanish Culture Type Collection (CECT). E. coli88

    JM109 (DE3) was used for tannase expression in pURI3 vector (18). 89


    Growth Conditions and Production of Recombinant Tannase 91

    L. plantarum was grown in MRS medium at 30º C. E. coli strains were cultured in Luria-92

    Bertani (LB) medium at 37 ºC and 200 rpm. When required, ampicillin was added to 93

    the medium at a concentration of 100,μg/mL. The cloning and expression of the gene 94

    encoding the L. plantarum CECT 748T tannase, tanLp1 (named lp_2956 in the L. 95

    plantarum WCFS1 strain) was previously described (18, 22 ). The growing of E.coli 96

    cells, their centrifugation and their disruption in order to obtain a of a crude protein 97

    extract were previously described (18). The crude tannase extract contained 8 mg of 98

    protein per mL. 99


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    Purification of tannase by adsorption on lowly activated Ni-IDA- 6% agarose gels. 101

    Lowly activated Ni-IDA-agarose gels ( containing 10 µEqs of chelates per mL of 6% 102

    agarose) were prepared as previously described (23). The crude tannase extract was 103

    diluted 10-fold in 50mM sodium phosphate buffer containg 150mM NaCl and 20 mM 104

    of imidazole and adjusted at pH 7.0. 150 mM NaCl was added to the binding buffer to 105

    prevent unspecific ionic interactions between non-recombinant proteins and the 106

    support. 20 mM imidazol was used in order to minimize the adsorption of non-107

    recombinant proteins on the lowly activated Ni-IDA-supports. 50 mL of the diluted 108

    crude tannase extract (0.8 mg/mL of protein concentration) were mixed with 1mL of 109

    lowly activated Ni-IDA-agarose support (23). The incubation was carried out at 25 ºC 110

    and under constant gentle magnetic stirring. After 1 h the enzyme was completely 111

    adsorbed on the chromatographic support. Then, the adsorbed enzyme was 112

    recovered by filtration and subsequently washed with 50 mL of 50 mM phosphate 113

    buffer pH 7.0 containing 50 mM imidazole and 150 mM NaCl in order to remove the 114

    traces of non-recombinant proteins adsorbed on the support. Finally, the desorption 115

    of tannase was performed by incubation of the chromatographic support for 30 min. 116

    with 50 mL of 50 mM phosphate buffer pH 7.0 containing 100 mM imidazole and 150 117

    mM NaCl. 118





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    Protein determination and enzymatic assays. 123

    Protein concentrations were determined by the Bradford´s method (24). Bovine serum 124

    albumin (BSA) was used as the standard. The esterase activity of tannase was 125

    determined using a rhodamine assay specific for gallic acid (25). A tannase activity unit 126

    was defined as the amount of enzyme needed to hydrolyze 1 µmol of methyl gallate 127

    per minute. 128


    SDS–PAGE analysis 130

    SDS–PAGE experiments were performed as described by Laemmli (26) in a SE 250-131

    Mighty small II electrophoretic unit (Hoefer Co. San Francisco, USA) 132

    (www.hoeferinc.com) using gels of 12% polyacrylamide in a separation zone of 9 cm ×133

    6 cm and a concentration zone of 5% polyacrylamide. Gels were stained with the 134

    Coomassie brilliant blue (R-250) method. Low-molecular mass marker kits from 135

    Pharmacia were used (Mr = 14 000–94 000). 136


    Immobilization of Tannase on CNBr-activated support 138

    The immobilization on CNBr-activated support was carried out at pH 7, 4º C for 15 min 139

    in order to strongly reduce the possibilities of a multipoint covalent attachment. 5 g of 140

    CNBr-activated support was added to a solution of 50 mL of purified tannase 141

    preparation. After 15 min, around 30% of enzyme was immobilized on the support. 142

    The immobilization process was ended by incubating the support with 1 M 143


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    ethanolamine at pH 8 for 2 h. Finally, the immobilized preparation was washed with 144

    abundant water. 145

    Immobilization of tannase on glyoxyl-agarose supports 146


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