Ó Springer 2006
World Journal of Microbiology & Biotechnology (2006) 22: 595–602 DOI 10.1007/s11274-005-9076-2
Transglutaminase-catalysed glycosidation of trypsin with aminated polysaccharides Maria L. Villalonga1, Reynaldo Villalonga1,*, Loredana Mariniello2, Leissy Go´mez1, Prospero Di Pierro1 and Raffaele Porta2 1 Enzyme Technology Group, University of Matanzas, Autopista a Varadero km 3 1/2, 44740 Matanzas, C.P., Cuba 2 Dipartimento di Scienza degli Alimenti, Universita’ di Napoli ‘‘Federico II’’, Via Universita’ 100, Portici, Naples, Italy *Author for correspondence: Tel.: +53-45-261251, Fax: +53-45-253101, E-mail:
[email protected] Received 15 June 2005; accepted 29 September 2005
Keywords: Enzyme thermostability, modified enzyme, polysaccharide, transglutaminase, trypsin
Summary Dextran (MW=7.2104), carboxymethylcellulose (MW=2.5104, substitution degree=0.7) and Ficoll (MW=6.9104) were derivatized with 1,4-diaminobutane and covalently attached to bovine pancreatic trypsin through a transglutaminase-catalysed reaction. The conjugates contained an average of 0.7–1.8 mol of polymers per mol of protein, and retained about 61–82% of the original esterolytic activity of trypsin. The optimum pH for trypsin was shifted to alkaline values after enzymatic glycosidation. The thermostability of the polymer–enzyme complexes was increased in about 13.7–50.0 °C over 10 min incubation. The prepared conjugates were also more stable against thermal incubation at different temperatures ranging from 50 °C to 60 °C. In comparison with native trypsin, the enzyme-polymer complexes were about 22- to 48-fold more resistant to autolytic degradation at pH 9.0. Transglutaminase-catalysed glycosidation also protected trypsin against denaturation in surfactant media, with 9- to 68–fold increased half-life times in the presence of 0.3% (w/v) sodium dodecylsulfate.
Introduction Industrial application of enzymes is often limited by the low stability properties of these biocatalysts under common technological conditions. Manipulation of the protein surface by covalent modification with watersoluble polymers constitutes the most effective stabilization strategy when the enzymes are required to digest insoluble or macromolecular substrates, or when the diffusion-determining reaction rate in the homogeneous state is higher than that in the heterogeneous state (Chen & Hsu 1997). During recent years, carbohydrate polymers have been extensively used as modifiers for stabilizing enzymes (Sundaram & Venkatesh 1998; Villalonga et al. 2000; Masa´rova´ et al. 2001; Villalonga et al. 2003a). This selection has been inspired by naturally occurring glycoenzymes, in which the sugar residues play an important role in the stability properties of these proteins (Wang et al. 1996). In this regard, the covalent attachment of ionic and non-ionic polysaccharides such as chitosan (Go´mez et al. 2000; Darias & Villalonga 2001), pectin (Go´mez & Villalonga 2000), dextran (Blomhoff & Christensen 1983; Srivastava 1991),
polymerized sucrose (Sundaram & Venkatesh 1998; Venkatesh & Sundaram 1998), carboxymethylcellulose (Villalonga et al. 1999; Ramı´ rez et al. 2002), mannan (Masa´rova´ et al. 2001), alginate (Go´mez et al. 2001) and cyclodextrin-grafted polysaccharides (Darias et al. 2002; Villalonga et al. 2003a) has been reported as a useful tool for increasing stability of enzymes. In general, the preparation of enzyme–polysaccharide conjugates has been accomplished through chemical procedures. However, enzyme-catalysed processes have several advantages when comparing with chemical ones, such as mild reaction conditions and low toxicity. In addition, the application of enzymes in neoglycoconjugate synthesis could allow the use of non-classical amino acid residues as modification points and the possibility of continuous production of conjugates using immobilized enzymes. As part of our interest in developing nonchemical methods for preparing neoglycoenzymes, we recently described the novel use of the enzyme transglutaminase (TGase, EC 2.3.2.13) to synthesize several trypsin–cyclodextrin conjugates having noticeable stability against thermal and autolytic inactivation (Villalonga et al. 2003b,c). In this regard, the present work represents an extension of our previous work that
596 provides a broadening of the proposed synthetic strategy. TGase catalyses the formation of intra- and intermolecular isopeptide cross-links between the c-carboxamide groups of endoprotein glutamine residues as acyl donor substrate and the e-amino groups of endoprotein lysine residues as acyl acceptor (Aeschlimann & Paulsson 1994). In addition, other compounds containing primary amino groups can act as acyl acceptors substrates for TGase. This enzyme has been isolated and purified from several microorganisms in Japan, and is currently used in that country for modifying protein foods at industrial level (Ando et al. 1989; Kanaji et al. 1993; Washizu et al. 1994; Mizuno et al. 2000; Ohtsuka et al. 2000). TGase has been successfully used for modifying the biological properties of several proteins and peptides of pharmacological interest by covalently linking polyamines to their reactive endo-glutamine residues (Metafora et al. 1989; Porta et al. 1990; Persico et al. 1992; Esposito et al. 1995, 1999; Mancuso et al. 1996, 1998, 2001; Tufano et al. 1996). Furthermore, TGase has been recently employed as a biotechnological tool for preparing edible films from pectin and soy flour (Mariniello et al. 2003). The present paper deals with the use of aminated derivatives of dextran (DEX), carboxymethylcellulose (CMC) and Ficoll (FIC) as acyl TGase acceptors to modify bovine pancreatic trypsin (EC 3.4.21.4), as well as the effect of this transformation on the catalytic and the stability properties of the glycosidated enzymes.
Materials and methods Materials Bovine pancreatic trypsin, N-a-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) and Fractogel EMD BioSEC (S) were obtained from Merck (Darmstadt, Germany). TGase (8 U/mg based on the hydroxamate activity assay, Folk & Chung 1985) from Streptoverticillium sp. was obtained from Ajinomoto Co. (Japan) and used as received. Polymerized sucrose (Ficoll 70, FIC, MW=6.9104) and dextran (MW=7.2104) were purchased from Pharmacia Biotech (Uppsala, Sweden). Low viscosity carboxymethylcellulose sodium salt (CMC, MW=2.5104, substitution degree=0.7) was obtained from BDH (Poole, UK). All other chemicals were analytical grade.
M. L. Villalonga et al. Diaminobutane (10 mmol) was further added to the activated polymer solutions, and then treated with 20 mg of NaBH4 for 4 h under continuous stirring. The modified polymer solutions were dialysed against distilled H2O and finally lyophilized. The molecular weights of the native polysaccharides were determined by analytical gel permeation chromatography on TESEK Hema-bio columns 40, 100, 300 and 1000 (430 cm) calibrated with pullulan standards. The degree of carboxymethylation of CMC was determined by 1H-NMR spectrometry (Ho & Klosiewics 1980) using a Bruker AVANTE 500 MHz apparatus. The degree of modification for the polymers was determined by measuring the amount of free amino groups with o-phthalaldehyde using glycine as standard (Bruneel & Schacht 1993). TGase-catalysed synthesis of trypsin-polymer conjugates TGase (100 lg protein) was added to reaction mixtures containing 10 mg of trypsin and 25 mg of each aminated polysaccharides, dissolved in 5 ml of 50 mM sodium phosphate buffer, pH 6.0. The reaction was stirred for 1 h at room temperature and for 3 h at 4 °C, and then applied to a gel filtration column Fractogel EMD BioSEC (S) (2.660 cm), equilibrated with 100 mM NaCl in 20 mM sodium acetate buffer, pH 5.0. The fractions containing the polymer-trypsin conjugates were pooled and kept at 4 °C. Assays Esterolytic activity of native and modified trypsins was determined at 25 °C in 67 mM Tris–HCl buffer, pH 8.0 using BAEE as substrate (Schwert & Takenaka 1955). One unit of esterolytic activity is defined as the amount of enzyme that hydrolyses 1.0 lmol of BAEE per minute at 25 °C. Michaelis–Menten parameters were calculated from Eadie-Hofstee plots. Proteolytic activity was determined using milk casein as substrate (Laskowski 1955). One unit of proteolytic activity, katal, is defined as the amount of enzyme that releases one mole of tyrosine per second at 25 °C. Total carbohydrates were determined by the phenolsulphuric acid method (Dubois et al. 1956) using glucose as standard. Protein concentration was estimated by the Lowry method using bovine serum albumin as standard. The conjugates prepared were analysed by 12.5% SDS/PAGE by the method reported by Laemmli (1970), visualizing the gel by Coomassie staining.
Synthesis of the modifying polymers pH optimum Polysaccharides were oxidized by dissolving 100 mg of the polymers in 5 ml of H2O and treated with 400 mg of sodium metaperiodate under continuous stirring at 4 °C in the dark during 2 h. The oxidation reaction was stopped by adding 100 ll of ethylene glycol and stirred for 1 h, and further dialysed against distilled H2O. 1,4-
The enzyme activities of native and polymer-modified trypsin preparations (20 lg ml)1) were measured at 25 °C Vs 3 mM BAEE in different buffer solutions: 50 mM sodium acetate, pH 5.0–6.0, 50 mM sodium phosphate, pH 6.5–8.0 and 50 mM sodium borate, pH 8.5–12.
Transglutaminase glycosidation of trypsin Thermostability Native and modified enzyme preparations (50 lg ml)1) were incubated at different temperatures in 20 mM sodium acetate buffer, pH 5.0. Aliquots were removed after 10 min incubation, diluted in cold 100 mM Tris– HCl buffer, pH 8.0, and assayed for esterolytic activity. Kinetics of thermal inactivation Native and conjugated trypsins (50 lg ml)1) were incubated at 50, 55 and 60 °C in 50 mM sodium acetate buffer, pH 5.0. Aliquots were removed at scheduled times, chilled quickly, and assayed for enzymatic activity as described in section 2.4. The half-life times were calculated from the first-order rate constants, ki, of inactivation, obtained from linear regression in logarithmic coordinates. Autolysis Native and modified trypsin forms (50 lg ml)1) were incubated at 35 °C in 50 mM Tris–HCl buffer, pH 9.0. Aliquots were removed at different times, diluted in 100 mM sodium acetate buffer, pH 5.0 and further assayed for esterolytic activity. Stability in the presence of sodium dodecylsulfate (SDS) Native and CMPCD-modified enzyme preparations (50 lg ml)1) were incubated at 30 °C in 0.3% SDS in 0.1 M Tris–HCl buffer, pH 9.0. Aliquots were removed at scheduled times, diluted in 0.1 M Tris–HCl buffer, pH 8.0, and assayed for esterolytic activity.
Results For preparing polymer derivatives suitable for modifying trypsin via a TGase-catalysed reaction, the polysaccharides were previously oxidized with sodium metaperiodate to generate aldehyde groups in the polymer chains (Scheme 1). The activated polysaccharides were further treated with 1,4-diaminobutane in the presence of NaBH4. Amination of the polymers resulted in the incorporation of 50, 120 and 110 mol amino groups per mol of CMC, DEX and FIC, respectively. These aminated polysaccharides were finally attached to the reactive glutamine residues located at the protein surface of trypsin using Streptoverticillum sp. TGase as biocatalyst. The formation of these polymer–enzyme adducts was confirmed by SDS-PAGE, as is shown in Figure 1 for the CMC–trypsin conjugate. As can be seen, a low molecular weight band is observed in the lane corresponding to trypsin the protease is autolytically degraded when is incubated with the polysaccharide in the absence of TGase. In contrast, the molecular weight of trypsin was increased after enzymatic glycosidation with this polysaccharide without
597 significant autodegradation. It should be noted that a more complex electrophoretic pattern was obtained by trypsin modified with aminated DEX and FIC (data not shown), probably associated with a higher interaction of SDS with the positively charged polymers. Table 1 reports the structural and the catalytic properties of the neoglycoenzymes prepared. The carbohydrate content of the conjugated trypsins represented 65, 82 and 67% by weight of the CMC-, DEXand FIC-modified enzymes forms, respectively. According to the average molecular weight of the polysaccharides, this result represented an average of 1.8, 1.5 and 0.7 mol of CMC, DEX and FIC respectively attached to each mole of enzyme. Trypsin retained about 33–60% of its proteolytic activity after enzymatic glycosidation with the polysaccharides. Toward a small substrate, BAEE, the specific activity of the modified enzymes represented 61–82% of that of the native trypsin. The TGase-mediated incorporation of DEX and FIC not significantly affected the affinity of trypsin for BAEE substrate On the contrary, the affinity of trypsin for BAEE was 2.4-fold increased after modification with aminated CMC. Figure 2 shows the effect of pH on the esterolytic activity of native and modified trypsin forms. The modified trypsins were significantly less sensitive to alkaline conditions than the native enzyme, shifting their ranges of maximal esterolytic activity to higher values of pH: the optimum pH of native trypsin lies in the range of 7–8, whereas the optimum pH of the modified enzyme covers the range 8–12. The thermoresistance of native and glycosidated trypsin forms was determined from the activity retained after heating the enzyme preparations at different temperatures for 10 min. Under these conditions, the thermostability of the polymer–protease complexes was remarkably improved, as shown in Figure 3. In this regard, the resistance to incubation at temperatures higher than 45 °C was increased for the conjugates prepared. The calculated value for T50, defined as the temperature at which 50% of the initial activity was retained, was increased from 49.5 °C to 63.2 °C and 66.5 °C for trypsin after modification with CMC and FIC, respectively. A noticeable thermostabilization was conferred to this protease after TGasemediated conjugation with DEX, as is revealed by the higher value of DT50=50 °C calculated for this neoglycoenzyme. The time course of thermal inactivation for nonmodified and polymer-modified trypsins was determined in the range of temperatures between 50 and 60 °C. Figure 4 shows that all enzyme forms lost activity progressively with time of incubation at each evaluated temperature, but the half-life times of modified enzymes were always higher than those of the corresponding to native trypsin (Table 2). It should be noted the lower thermal resistance conferred to this protease after glycosidation with CMC, in comparison with the DEX– and FIC–trypsin complexes.
598
M. L. Villalonga et al. O
O OH
HO HO
O
O OH
HO HO
O
O
(c)
O
OH
HO HO
O OCH2CO2Na
OH
HO HO
O
O
OH O
OH
O
O
HO
O
OH
HOHO
O
O
HO
O
(a)
OCH2CO2Na O
HO O
HO
OH
OH
O
OH
OCH2CO2Na
CMC (Sodium salt)
m-NaIO4
OCH2CO2Na
OCH2CO2Na O
O
O
O O
OCH2CO2Na O
HO
O O
O
O
O O
O
O O
O
HO
O OCH2CO2Na
OH HO OH
O
NH2
O
NaBH4
NH2
OH
HO
Polyaldehyde derivative of CMC
OH O
O
NH2 O
O HO HO
O
OH
HO
O O
O OH
OH
HO
O
OH
O O
HO
OCH2CO2Na
OCH2CO2Na OH
HN
O
OH
O
O O
O
OCH2CO2Na O
HO
O
OH
O
O
O
O O
HN
HN
HN
HO
O
OH
HO OH
HO
OH
HO
HO
O OCH2CO2Na
O O
O O
O
HO OH
O
HO
Aminated CMC
OH O O
H2N
NH2
H2N
(b) Trypsin-CONH2
TGase, pH 6.0
NH2
OCH2CO2Na
OCH2CO2Na HN
OH O
O O
OCH2CO2Na O
HO
O O
O O HN
O HN
HN
HO
OH
OCH2CO2Na
Trypsin-CMC H2N
NH
HN O O
TRYPSIN O
NH2
O
NH2
Scheme 1. Structure of DEX (a), FIC (b, proposed), and CMC (c). Synthesis of CMC–trypsin conjugate.
The time-course of autolytic inactivation of native and modified trypsins was studied by incubating all enzyme preparations at pH 9.0 and 30 °C. Under these conditions, the catalytic activity of native protease was completely lost after 90 min of incubation, as is shown in Figure 5. On the contrary, the CMC–, DEX– and FIC–trypsin complexes were markedly more resistant to autolytic inactivation, retaining about 54, 72 and 80%
of the initial activity after 3 h of incubation, respectively. The stability of the glycosidated trypsins in the presence of anionic surfactants was evaluated by incubating the enzymes in 0.3% SDS solutions at pH 9.0. As is illustrated in Figure 6, native trypsin was rapidly inactivated according to a first-order kinetic, with a half-life time of about 5 min. On the other hand, the resistance
599
Transglutaminase glycosidation of trypsin Table 2. Half-life times of native and polysaccharide-modified trypsins at different temperatures.a Half-life time (min)
Trypsin Trypsin–CMC Trypsin–DEX Trypsin–FIC
50 °C
55 °C
60 °C
15.6 44.1 315 295
7.9 35.0 210 198
5.5 29.4 154 150
a
Average of three measurements. Relative error
