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Thermal stability and conformational changes of transglutaminase from a newly isolated Streptomyces hygroscopicus
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 3794–3800
Thermal stability and conformational changes of transglutaminase from a newly isolated Streptomyces hygroscopicus Li Cui a
a,b
, Guocheng Du b, Dongxu Zhang b, Jian Chen
b,*
Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, Southern Yangtze University, Wuxi 214122, China b Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214122, China Received 24 May 2007; received in revised form 3 July 2007; accepted 4 July 2007 Available online 24 August 2007
Abstract Thermal stability and conformational changes of transglutaminase (TGase) from a newly isolated Streptomyces hygroscopicus were investigated in this study. The inactivation kinetics of the microbial transglutaminase (MTGase) was fitted using one-step inactivation model. It was much more stable under 40 C. The half-lives for the MTGase at 50 C and 60 C were only 20 min and 8 min, respectively. Spectroscopic studies of the enzyme suggested conformational transition from ordered secondary structural elements (a/b-protein) to unordered structure during thermal denaturation. Some polyols could improve the thermal stability of the enzyme. Among the polyols examined, the prolonged half-lives of 40 min at 50 C and 20 min at 60 C were gained by adding 10% glycerol. The results of differential scanning calorimetric (DSC) analysis showed a distinct transition peak with a significant greater Tm and DH for the MTGase mixed with polyols in comparison with the control, which indicated that the polyols could maintain the natural structure of the enzyme to some extent. The SDS–PAGE electrophoresis of cross-linked casein confirmed that the stabilizers could protect the MTGase from thermal denaturation. 2007 Elsevier Ltd. All rights reserved. Keywords: Microbial transglutaminase; Streptomyces hygroscopicus; Thermal stability; Conformational changes
1. Introduction Transglutaminase (TGase; protein-glutamine-glutamyltransferase, EC 2.3.2.13) is an enzyme capable of catalyzing acyl transfer reactions by introducing covalent cross-links between proteins as well as peptides and various primary amines. Transglutaminases present in most animal or vegetable tissues and body fluids are involved in several biological processes (Aeschlimann and Paulsson, 1994). In the recent years, TGase has gained interest in the view of its attractive potential application in food industries (Zhu et al., 1995; Motoki and Seguro, 1994), immobilization of enzymes (Yoshiro et al., 1992; Josten et al., 1999) and tex-
*
Corresponding author. Tel.: +86 510 85913661; fax: +86 510 85888301. E-mail address: [email protected] (J. Chen). 0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.07.017
tile industries (Cortez et al., 2004). The relatively small quantity obtained and the complex separation and purification procedure required for these enzymes from tissues led to the search for microbial sources. Ando et al. (1989) first reported that strains from the genus Streptoverticillium screened from several thousands microorganisms had the ability to produce transglutaminase using the hydroxamate assay. These microorganisms excreted the enzyme, and one of them classified as a variant of Streptoverticillium mobaraensis (Washizu et al., 1994) produced a high activity. The enzyme from microorganisms was named microbial transglutaminase (MTGase). Since then, efforts have been made to obtain TGase not only from Streptoverticillium species but also from other genus such as Bacillus (Zhu et al., 1996; Barros et al., 2003). To achieve commercialization of MTGase, studies have been continually performed in our laboratory including optimization of cultivation conditions, downstream process
L. Cui et al. / Bioresource Technology 99 (2008) 3794–3800
optimization and its application (Zheng et al., 2002; Yan et al., 2005; Cui et al., 2006; Du et al., 2007). We have reported a newly isolated strain classified as Streptomyces hygroscopicus which produced a high TGase activity. The characterizations of the enzyme made it a good candidate for application in food industries (Cui et al., 2007). The use of the enzyme for industrial purposes needs depend on its stability during preparation, storage and application under some harsh conditions. For example, it should be stable and active at high temperature or in organic solvents (Cowan, 1997; Daniel, 1982). Thus, the objective of this report was to study the thermal stability of TGase from the newly isolated strain. Furthermore, the conformational changes induced by temperature were established by means of ultraviolet absorption, fluorescence spectroscopic and far-UV circular dichroism (CD) measurements. In order to improve the thermal stability of the enzyme, some stabilizers were used and their protection effect was estimated with differential scanning calorimeter (DSC) and sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS– PAGE). Investigation of this paper will help to determine the suitability of this enzyme for commercial preparation and industrial applications. 2. Methods 2.1. Materials All the chemicals used were of analytical grade and mainly purchased from Sinopharm Chemical Reagent Co., Ltd, China, unless otherwise mentioned. N-carboxybenzoyl-L-glutaminyl-glycine (N-CBZ-Gln-Gly) was purchased from Sigma Chemical Co., Ltd. 2.2. Enzyme preparation MTGase from Streptomyces hygroscopicus WSH03-13 was purified from the culture medium as described previously (Cui et al., 2007). 2.3. Determination of enzyme activity MTGase activity was determined by hydroxamate formation with the specific substrate, N-CBZ-Gln-Gly described by Grossowicz et al. (1950). One unit of transglutaminase was defined as the amount of enzyme which causes the formation of 1.0 lmol L-glutamic acid c-monohydroxamate per minute at 37 C. 2.4. Thermal stability test Thermal stability of the MTGase was studied at pH 6.0 in 20 mmol/L Tris–HCl buffer with an enzyme activity of 1.0 U/mL at different temperature of 20, 30, 40, 50, 60 C. The samples were incubated for appropriate periods of time, aliquots were then withdrawn, and the residual
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activities of the enzyme were determined by using the method described above, taking the activity of a sample without incubation as 100%. The effect of some polyols on thermal stability of the enzyme was examined by incubating the enzyme preparation at 50 C for 30 min in the presence of polyethylene glycol 6000 (PEG) at a concentration of 0.8 mol/L or with 10% (w/v) sorbitol, glycerol, sucrose, fucose and maltodextrin, respectively. Residual enzyme activity was measured. All experiments were conducted in triplicate, results shown are mean values. 2.5. Ultraviolet spectra, fluorescence emission spectra, and circular dichroism spectra measurements Ultraviolet spectra were recorded from 220 nm to 300 nm with a Shimadzu UV2450 spectrophotometer. The fluorescence spectra were measured with a Hitachi 650-60 spectrofluorometer. The excitation wavelength was 295 nm. A Jasco J715 CD spectropolarimeter was used for CD measurements in the far-ultraviolet region from 190 to 250 nm. The sample cell path length was 1 mm. Five spectra were accumulated and averaged for each sample. Mean residue ellipticity, [h]MR, was expressed in deg cm2 dmol1, using a mean residue weight of 110. Percentages of secondary structures were calculated by applying the Sreerama and Woody method (Sreerama and Woody, 1994). 2.6. Differential scanning calorimetry analysis Differential scanning calorimetry (DSC) analysis was carried on a Mettle 882 DSC (Mettle-Toledo, Switzerland). The samples for DSC measurements were scanned at a heating rate of 1 C /min from 20 to 90 C. The onset transition temperature (Tm) and calorimetric enthalpy (DH) were used as conformational stability indicating thermodynamic parameters. Increase in DH and Tm of the MTGase was interpreted as an indication of stabilizing effect provided by different polyols. All data manipulations were performed by using Origin software provided with the DSC. 2.7. Sodium dodecylsulfate–polyacrylamide gel electrophoresis The cross-linking reactions catalyzed by MTGase were carried out at 37 C in 20 mmol/L phosphate buffer (pH 6.5) containing 1% (w/v) casein as the substrate. The reaction mixtures were incubated for 10 min, then stopped by directly mixing with the sample buffer (2·) of electrophoresis and analyzed by SDS–PAGE according to the method of Laemmli (Laemmli, 1970). A 12.5% separating gel was used. The proteins were stained with a 0.1% solution of Coomasiie Brilliant Blue R-250.
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3. Results and discussion
100
3.1. Thermal stability of MTGase 80
Flourescence (%)
As was expected the thermal inactivation kinetics of the MTGase were fitted using one-step inactivation model according to Fig. 1. The inactivation rate of the enzyme was calculated by first-order expression: ln[Ut/U0] = kt. The k (inactivation rate constant or first-order rate constant) values were calculated from a plot of ln[Ut/U0] versus t at a particular temperature. The thermal inactivation of the enzyme at different temperature could be distinguished by the differences in the slopes determined by linear regression (Fig. 1). The transglutaminase produced by S. hygroscopicus was very stable below 40 C. The model parameters: inactivation rate constants (k, min1) and half-lives (t1/2, min) for MTGase at different temperatures (20 C, 30 C and 40 C) were 0.0009, 0.0016, 0.0072 and 770, 433, 96, respectively after calculation with the equations: ln[Ut/U0] = kt and t1/2 = ln2/k. When the temperature was above 50 C, the enzyme was inactivated rapidly and the inactivation rate constants (k, min1) and half-lives (t1/2, min) at 50 C and 60 C were 0.0350, 0.0828 and 20, 8, respectively.
40
0 min 20 min 40 min 60 min
20
0 300
20
30
Time (min) 60
100
-2
ln (Ut/U0)
-4
360
380
400
0.2
Absorbance
0.15
0.1
120
0
340
Fig. 2. Fluorescence emission spectra of MTGase after different periods of incubation at 60 C.
0.05
10
320
Wavelength (nm)
3.2. Conformational changes during heat treatment of the MTGase Changes in the fluorescence emission spectra of the MTGase after 0, 20, 40 and 60 min of incubation at 60 C were shown in Fig. 2. The fluorescence emission intensity markedly increased with prolonged heat treatment periods at 60 C and no obvious red or blue shift occurred to the fluorescence emission spectra. Fig. 3 showed the effect of heat treatment time on the ultraviolet spectra of the enzyme. The native MTGase exhibited an absorption peak at about 275 nm which was contributed
60
0 200
0 min 20 min 40 min 60 min 220
240
260
280
300
Wavelength (nm) Fig. 3. UV-absorbance spectra of MTGase after different periods of incubation at 60 C.
-6 -8 -10 -12
Fig. 1. Kinetics of thermal inactivation of MTGase from Streptomyces hygroscopicus. (4) 20 C: y = 0.0009x, R2 = 0.9886; (h) 30 C: y = 0.0016x, R2 = 0.9893; (s) 40 C: y = 0.0072x, R2 = 0.9937; (·) 50 C: y = 0.0350x, R2 = 0.9918; (–) 60 C: y = 0.0828x, R2 = 0.999.
by Tyr and Try residual in the protein molecule. The absorption significantly increased in magnitude after 20 min of incubation at 60 C. When the heat treatment time was prolonged further, the peak increased more slowly. No evident changes occurred of the absorption intensity for the enzyme treated at 60 C for 60 min by comparing with that for 40 min. This result was consistent with that of the fluorescence emission spectra. The spectral results indicated that the ordered structure was almost completely in an unfolded state after a 40-min incubation
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at 60 C and all the enzyme activity was lost simultaneously. Fig. 4 depicted the far-UV CD spectra of the MTGase from S. hygroscopicus when the temperature raised to 50 C and 60 C. The native MTGase from S. hygroscopicus was a a + b protein from the CD spectra with separate a-helix and b-sheet rich regions. The CD spectra of such proteins in water are dominated by the a-helix portion, exhibiting negative bands at around 208 and 222 nm and a positive band below 195 nm (Manavalan and Johnson, 1983). The MTGase contains about 20.5% a- helix and 33.1% b-sheet, which was quite similar to that from Streptoverticillium mobaraense (24.5% a-helix, 23.8% b-strand, 21.1% b-turn and 30.7% unordered structure) (Mene´ndez et al., 2006). With the temperature raised to 50 C, the intensities of the positive band and the negative CD bands decreased, without significant changes in the spectral form and marked wavelength shifts. The typical a-helix structure still retained and Sreerama and Woody method yielded 17.5% a-helix and 32.4% b-strand (Table 1). The unordered structure content increased from 46.6% to 50.1%. As the temperature increased further to 60 C, the intensity of the negative peaks at 208 nm and 220 nm decreased significantly and a pronounced blue shift at 208 nm was observed on the CD spectra. Analysis of the CD spectra
Fig. 4. Far-UV CD spectra of MTGase at different temperature.
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Table 1 Secondary structure and relative activity of MTGase at different temperatures Temperature (C)
a-Helix (%)
b-Sheet (%)
Turn + coil (%)
Relative activity (%)
25 50 60
20.5 17.5 12.5
33.1 32.4 28.9
46.6 50.1 58.5
100 60 20
of the denatured MTGase revealed that some of the secondary structure elements of this enzyme persisted even under such condition. The a-helix structure content of the enzyme decreased to 12.5% and the unordered structure content increased to 58.5% continually. Because it should take some time for the enzyme to unfold the natural structure, even at 60 C, 20% relative activity was still detected by using the activity determination method described above (using the activity of a sample without incubation as 100%). 3.3. Effect of polyols on thermal inactivation of MTGase Polyols have long been regarded as protein stabilizers (Hafedh et al., 2001). To examine the protective effect of some stabilizers on thermal inactivation of MTGase, the residual activity was determined after incubation at 50 C for 30 min in the presence of PEG, sorbitol, glycerol, sucrose, fucose and maltodextrin, respectively. As shown in Fig. 5, after a 30-min incubation at 50 C, about 70% activity of the enzyme was lost. At the given concentrations of different stabilizers, glycerol and sorbitol exhibited a higher stabilizing effect and about 75% activity of the enzyme was preserved. The nonpolar polyol PEG also played the same role as maltodextrin in protecting MTGase from heat denaturation at 50 C. The apparent halflives of the enzyme increased to 38 and 44 min approximately at 50 C by adding PEG and glycerol, respectively when compared to the control sample without any stabilizers (t1/2, 20 min) after calculation by using the first-order
Fig. 5. Effect of different stabilizers on the residual activity of MTGase after 30 min of incubation at 50 C with PEG at a concentration of 0.8 mol/L and with 10% (v/v) sorbitol, glycerol, sucrose, fucose and maltodextrin, respectively.
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expression: ln[Ut/U0] = kt (Fig. 6). Even the incubation temperature raised to 60 C, PEG and glycerol enhanced the thermal stability of the enzyme and the half-lives increased from 8 min to 16 and 21 min, respectively (Fig. 6). The improvement of thermal stability by adding polyols was probably due to the reinforcement of the hydrophobic interactions among nonpolar amino acids inside the enzyme molecules, and thus increased their resistance to inactivation, since it had been reported that polyhydroxy compounds modify the structure of water and/or strengthen hydrophobic interactions among nonpolar amino acids inside the protein molecules (Back et al., 1979). Moreover, PEG is a nonpolar polyol and can evidently increase the viscosity of the enzyme solution, which may cause reduction of chemical or biological reaction rate which could result in inactivation of the enzyme (Lee and Choo, 1989). 3.4. Analysis of protective effect of polyols by DSC and SDS–PAGE DSC is ideally suited to the study of protein thermal denaturation in solution since it measures the forces stabilizing the conformational structure directly and is therefore model independent (Richard et al., 1998). Thermograms obtained by DSC for MTGase without any additives presented one main peak. This peak was an endothermic one which occurred at a temperature of 60.166 C which was defined as the onset transition temperature (Tm) of the folding/unfolding transition to the MTGase. The DH value for the transition was 4.5008 ± 0.02 J/g (Table 2). No peaks were visible on the rescan, which means that all tranTime (min) 30
60
90
120
0
-1
ln(Ut/U0)
-2
-3
-4
-5
-6
Fig. 6. Kinetics of thermal inactivation of MTGase from Streptomyces hygroscopicus in the presence of glycerol and PEG at 50 C (—) and 60 C (—). (d): control; (4): glycerol; (h): PEG.
Table 2 Effect of different stabilizers on onset transition temperature and calorimetric enthalpy of MTGase Polyols
Transition temperature, Tm (C)
Calorimetric enthalpy, DH (J/ g)
Control Sucrose Sorbitol Glycerol
60.166 ± 0.3 66.257 ± 0.2 64.981 ± 0.3 68.346 ± 0.6
4.5008 ± 0.02 4.5760 ± 0.02 4.7003 ± 0.03 4.8001 ± 0.05
sitions were irreversible after heating to 90 C. The effects of polyols on the conformational stability of the enzyme against heat treatment were deduced from the shift of this transition peak and the values for Tm and DH were shown in Table 2. It was clear that all of the polyols examined caused a higher Tm and a significant increase in DH value in comparison with the control. The Tm and DH values of the MTGase containing glycerol increased to 68.346 ± 0.6 C and 4.8001 ± 0.05 J/g, which indicated that glycerol exhibited a higher stabilizing effect. The presence of sucrose and sorbitol also enhanced the Tm and DH values of the enzyme to 66.257 ± 0.2 C, 64.981 ± 0.3 C and 4.5760 ± 0.02 J/g, 4.7003 ± 0.03 J/g, respectively. In addition, the DSC thermograms of the MTGase in the absence (control) and presence of the above three stabilizers were taken after 50 min of incubation at 60 C and chill immediately on ice (the thermograms not shown here). The MTGase without any additives did not show any transition peaks, which might be because of irreversible thermal denaturation of the enzyme. The enzyme mixed with polyols still showed a distinct transition peak with a significantly greater Tm values in comparison with the control. The results indicated that the polyols maintained the ordered structure to some degree during thermal unfolding of the enzyme. Glycerol was taken as an example for estimate of the protective effect of the polyols on MTGase against thermal inactivation by SDS–PAGE analysis. The enzyme used was pre-incubated at 60 C for different periods, aliquots were then withdrawn and chilled immediately. A casein-based system was used and the SDS-gel electrophoretic patterns obtained after cross-linking with MTGase. On the SDS– PAGE pattern, new protein biopolymers cross-linked by transglutaminase can congregate at the interface of the stacking and separating gel or stop at the upper part of the separating gel due to the increase of molecular weight. The cross-linking reaction conditions of all the samples: the reaction temperature, reaction time, pH, protein concentration and the enzyme amount were the same except for the enzyme containing glycerol in samples d, e, f and without glycerol in samples a, b, c. (Fig. 7). Lane a indicated the crosslinked products catalyzed by MTGase not containing glycerol after incubation for 10 min. There were some biopolymers at the interface of the stacking and separating gel and more new high molecular weight proteins appeared at the upper part of the separating gel by comparing with lane o, which suggested that
L. Cui et al. / Bioresource Technology 99 (2008) 3794–3800
Fig. 7. SDS–PAGE analysis of cross-linked casein at 37 C for 10 min by MTGase after different periods of incubation at 60 C. (s: casein solution without MTG; a, b, c: casein solution with MTG after incubation of 10 min. 20 min, 30 min at 60 C ; d, e, f: casein solution with MTGase containing 10% glycerol after incubation of 10 min. 20 min, 30 min at 60 C).
cross-linking reaction occurred because of the residual enzyme activity. Lane d showed the cross-linked products catalyzed by MTGase with 10% glycerol after incubation for 10 min. By comparing with lane a, there were much more biopolymers on the top of separating and stacking gel and the amount of low molecular weight proteins decreased at the same time. The results indicated that more enzyme activity was retained by adding glycerol and enhanced the cross-linking reaction efficiency. Lane e showed the products catalyzed by MTGase containing glycerol after 20 min of incubation. The amount of the biopolymers induced by MTGase was much less than that in lanes a and d, which suggested that the majority of the enzyme activity was lost after incubation for 20 min despite the addition of glycerol. In the absence of glycerol, the extent of crosslinking of casein catalyzed by the MTGase was remarkably reduced as a function of pre-incubation time of the enzyme, and nearly completely inactivated after incubation for 30 min at 60 C (lane c). However, cross-linking of casein was considerably improved by adding glycerol during the pre-incubation (lanes d, e and f) and more new high MW biopolymers increased on the top of separating and stacking gel by comparing with the MTGase without glycerol. The results showed that glycerol was involved in the stabilization of MTGase by reinforcing the hydrophobic interactions among nonpolar amino acids inside the protein molecules, and thus rendering them resistant to thermal inactivation mentioned above (Back et al., 1979). 4. Conclusions TGase has captured peoples’ intense interest due to its attractive potential application for its special catalytic character. It is very important to obtain the enzyme with high thermal stability and activity for its preparation and application in industrial purposes. Considering this fact, we report here the thermal stability and conformational
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changes of transglutaminase produced by a newly isolated strain S. hygroscopicus. The enzyme was stable under 40 C. Some polyols: sorbitol, sucrose, fucose, maltodextrin, polyethylene glycol 6000 (PEG) and glycerol, could improve the thermal stability of the enzyme. The half-lives for the MTGase at 50 C and 60 C were increased from 20 min and 8 min to 40 min and 20 min, respectively by mixing with 10% glycerol, Spectroscopic studies of the MTGase indicated conformational transition from ordered secondary structural elements (a/b protein) to unordered structures during thermal treatment. DSC and SDS–PAGE analysis of the cross-linked products by MTGase confirmed the protective effect of polyols on the enzyme against thermal denaturation. The MTGase mixed with these polyols showed a distinct transition peak with a significant increase in Tm and DH values by comparing with the control. From the results above, the possibility of stabilizing the MTGase against thermal inactivation in the presence of polyols would be of great interest for industrial preparation and storage, which is important to make this enzyme a good candidate for application in different industries. Acknowledgements This project was financially supported by the Open Project Program of Key Laboratory of Eco-Textiles (Southern Yangtze University, China, No. KLET0623), program for New Century Excellent Talents in University and a grant from National Science Fund for Distinguished Young Scholars. References Aeschlimann, D., Paulsson, M., 1994. Transglutaminase: protein crosslinking enzymes in tissues and body fluids. Thromb. Haemostasis 71, 402–415. Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H., Motoki, M., 1989. Purification and characterization of a novel transglutaminase derived from micro-organisms. Agric. Biol. Chem. 53, 2613–2617. Back, J.F., Oakenfull, D., Smith, M.B., 1979. Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry 18, 5191–5196. Barros, S.L.H., Assmann, F., Zachia, A.M.A., 2003. Purification and Properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment. Biotechnol. Appl. Biochem. 37, 295–299. Cortez, J., Bonner, P.L.R., Griffin, M., 2004. Application of transglutaminase in the modification of wool textile. Enzyme Microb. Technol. 34, 64–72. Cowan, D.A., 1997. Thermophilic proteins: stability and function in aqueous and organic solvents. Comp. Biochem. Physiol. A Physiol 118, 429–438. Cui, L., Zhang, D., Huang, L., Liu, H., Du, G., Chen, J., 2006. Stabilization of a new microbial transglutaminase from Streptomyces hygroscopicus WSH03 – 13 by spray drying. Process Biochem. 41, 1427–1431. Cui, L., Du, G., Zhang, D., Liu, H., Chen, J., 2007. Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus. Food Chem., doi:10.1016/j.foodchem.2007.04.020.
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Daniel, R.M., 1982. A correlation between protein thermostability and resistance to proteolysis. Biochem. J. 207, 641–644. Du, G., Cui, L., Zhu, Y., Chen, J., 2007. Improvement of shrink-resistance and tensile strength of wool fabric treated with a novel microbial transglutaminase from Streptomyces hygroscopicus. Enzyme Microb. Technol. 40, 1753–1757. Grossowicz, N., Wainfan, E., Borek, E., Waelsch, H., 1950. The enzymatic formation of hydroxamic acids from glutamine. J. Biol. Chem. 187, 111–125. Hafedh, B., Semia, D.C., Ali, G., 2001. Stabilization of Penicillium occitanis cellulases by spray drying in the presence of maltodextrin. Enzyme Microb. Technol. 28, 253–258. Josten, A., Meuse, M., Spener, F., Haalck, L., 1999. Enzyme immobilization via microbial transglutaminase: a method for the generation of stable sensing surfaces. J. Mol. Catal. B: Enzym. 7, 57–66. Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of head of bacteriophage T4. Nature (London) 227, 680–685. Lee, Y.K., Choo, C.L., 1989. The kinetics and mechanism of shear inactivation of lipase from Candida cylindracea. Biotechn. Bioeng. 33, 183–190. Manavalan, P., Johnson Jr., W.C., 1983. Sensitivity of circular dichroism to protein tertiary structure class. Nature 305, 831–832. Mene´ndez, O.H., Rawel, Schwarzenbolz U., Henle, T., 2006. Structural changes of microbial transglutaminase during thermal and highpressure treatment. J. Agric. Food Chem. 54, 1716–1721. Motoki, M., Seguro, K., 1994. Trends in Japanese soy protein research. Information 5, 308–313. Richard, L., Remmele Jr., R.L., Nancy, S.N., Subhashini, S., Wayne, R.G., 1998. Interleukin-1 receptor (IL-1R) liquid formulation devel-
opment using differential scanning calorimetry. Pharm. Res. 15, 200– 208. Sreerama, N., Woody, R.W., 1994. Protein secondary structure from circular dichroism spectroscopy. Combining variable selection principle and cluster analysis with neural network, ridge regression and selfconsistent methods. J. Mol. Biol. 242, 497–507. Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Akagi, H., Motoki, M., Takeuchi, K., 1994. Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans. Biosci. Biotechnol. Biochem. 58, 82–87. Yan, G.L., Du, G.C., Li, Y., Chen, J., 2005. Enhancement of microbial transglutaminase production by Streptoverticillium mobaraense: application of a two-stage agitation speed control strategy. Process Biochem. 40, 963–968. Yoshiro, K., Erika, O., Masao, M., 1992. Enzyme immobilization on ion exchangers by forming an enzyme coating with transglutaminase as a crosslinker. Biosci. Biotechnol. Biochem. 56, 1323–1324. Zheng, M.Y., Du, G.C., Chen, J., 2002. pH control strategy of batch microbial transglutaminase production with Streptoverticillium mobaraense. Enzyme Microb. Technol. 31, 477–481. Zhu, Y., Rinzema, A., Tramper, J., Bol, J., 1995. Microbial transglutaminase: a review on its production and application in food processing. Appl. Microbiol. Biotechnol. 44, 277–282. Zhu, Y., Rinzema, A., Tramper, J., Bol, J., 1996. Medium design based on stoiciometric analysis of microbial transglutaminase production by Streptoverticillium mobaraense. Biotechnol. Bioeng. 50, 291–298.
Bioresource Technology 99 (2008) 3794–3800
Thermal stability and conformational changes of transglutaminase from a newly isolated Streptomyces hygroscopicus Li Cui a
a,b
, Guocheng Du b, Dongxu Zhang b, Jian Chen
b,*
Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, Southern Yangtze University, Wuxi 214122, China b Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214122, China Received 24 May 2007; received in revised form 3 July 2007; accepted 4 July 2007 Available online 24 August 2007
Abstract Thermal stability and conformational changes of transglutaminase (TGase) from a newly isolated Streptomyces hygroscopicus were investigated in this study. The inactivation kinetics of the microbial transglutaminase (MTGase) was fitted using one-step inactivation model. It was much more stable under 40 C. The half-lives for the MTGase at 50 C and 60 C were only 20 min and 8 min, respectively. Spectroscopic studies of the enzyme suggested conformational transition from ordered secondary structural elements (a/b-protein) to unordered structure during thermal denaturation. Some polyols could improve the thermal stability of the enzyme. Among the polyols examined, the prolonged half-lives of 40 min at 50 C and 20 min at 60 C were gained by adding 10% glycerol. The results of differential scanning calorimetric (DSC) analysis showed a distinct transition peak with a significant greater Tm and DH for the MTGase mixed with polyols in comparison with the control, which indicated that the polyols could maintain the natural structure of the enzyme to some extent. The SDS–PAGE electrophoresis of cross-linked casein confirmed that the stabilizers could protect the MTGase from thermal denaturation. 2007 Elsevier Ltd. All rights reserved. Keywords: Microbial transglutaminase; Streptomyces hygroscopicus; Thermal stability; Conformational changes
1. Introduction Transglutaminase (TGase; protein-glutamine-glutamyltransferase, EC 2.3.2.13) is an enzyme capable of catalyzing acyl transfer reactions by introducing covalent cross-links between proteins as well as peptides and various primary amines. Transglutaminases present in most animal or vegetable tissues and body fluids are involved in several biological processes (Aeschlimann and Paulsson, 1994). In the recent years, TGase has gained interest in the view of its attractive potential application in food industries (Zhu et al., 1995; Motoki and Seguro, 1994), immobilization of enzymes (Yoshiro et al., 1992; Josten et al., 1999) and tex-
*
Corresponding author. Tel.: +86 510 85913661; fax: +86 510 85888301. E-mail address: [email protected] (J. Chen). 0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.07.017
tile industries (Cortez et al., 2004). The relatively small quantity obtained and the complex separation and purification procedure required for these enzymes from tissues led to the search for microbial sources. Ando et al. (1989) first reported that strains from the genus Streptoverticillium screened from several thousands microorganisms had the ability to produce transglutaminase using the hydroxamate assay. These microorganisms excreted the enzyme, and one of them classified as a variant of Streptoverticillium mobaraensis (Washizu et al., 1994) produced a high activity. The enzyme from microorganisms was named microbial transglutaminase (MTGase). Since then, efforts have been made to obtain TGase not only from Streptoverticillium species but also from other genus such as Bacillus (Zhu et al., 1996; Barros et al., 2003). To achieve commercialization of MTGase, studies have been continually performed in our laboratory including optimization of cultivation conditions, downstream process
L. Cui et al. / Bioresource Technology 99 (2008) 3794–3800
optimization and its application (Zheng et al., 2002; Yan et al., 2005; Cui et al., 2006; Du et al., 2007). We have reported a newly isolated strain classified as Streptomyces hygroscopicus which produced a high TGase activity. The characterizations of the enzyme made it a good candidate for application in food industries (Cui et al., 2007). The use of the enzyme for industrial purposes needs depend on its stability during preparation, storage and application under some harsh conditions. For example, it should be stable and active at high temperature or in organic solvents (Cowan, 1997; Daniel, 1982). Thus, the objective of this report was to study the thermal stability of TGase from the newly isolated strain. Furthermore, the conformational changes induced by temperature were established by means of ultraviolet absorption, fluorescence spectroscopic and far-UV circular dichroism (CD) measurements. In order to improve the thermal stability of the enzyme, some stabilizers were used and their protection effect was estimated with differential scanning calorimeter (DSC) and sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS– PAGE). Investigation of this paper will help to determine the suitability of this enzyme for commercial preparation and industrial applications. 2. Methods 2.1. Materials All the chemicals used were of analytical grade and mainly purchased from Sinopharm Chemical Reagent Co., Ltd, China, unless otherwise mentioned. N-carboxybenzoyl-L-glutaminyl-glycine (N-CBZ-Gln-Gly) was purchased from Sigma Chemical Co., Ltd. 2.2. Enzyme preparation MTGase from Streptomyces hygroscopicus WSH03-13 was purified from the culture medium as described previously (Cui et al., 2007). 2.3. Determination of enzyme activity MTGase activity was determined by hydroxamate formation with the specific substrate, N-CBZ-Gln-Gly described by Grossowicz et al. (1950). One unit of transglutaminase was defined as the amount of enzyme which causes the formation of 1.0 lmol L-glutamic acid c-monohydroxamate per minute at 37 C. 2.4. Thermal stability test Thermal stability of the MTGase was studied at pH 6.0 in 20 mmol/L Tris–HCl buffer with an enzyme activity of 1.0 U/mL at different temperature of 20, 30, 40, 50, 60 C. The samples were incubated for appropriate periods of time, aliquots were then withdrawn, and the residual
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activities of the enzyme were determined by using the method described above, taking the activity of a sample without incubation as 100%. The effect of some polyols on thermal stability of the enzyme was examined by incubating the enzyme preparation at 50 C for 30 min in the presence of polyethylene glycol 6000 (PEG) at a concentration of 0.8 mol/L or with 10% (w/v) sorbitol, glycerol, sucrose, fucose and maltodextrin, respectively. Residual enzyme activity was measured. All experiments were conducted in triplicate, results shown are mean values. 2.5. Ultraviolet spectra, fluorescence emission spectra, and circular dichroism spectra measurements Ultraviolet spectra were recorded from 220 nm to 300 nm with a Shimadzu UV2450 spectrophotometer. The fluorescence spectra were measured with a Hitachi 650-60 spectrofluorometer. The excitation wavelength was 295 nm. A Jasco J715 CD spectropolarimeter was used for CD measurements in the far-ultraviolet region from 190 to 250 nm. The sample cell path length was 1 mm. Five spectra were accumulated and averaged for each sample. Mean residue ellipticity, [h]MR, was expressed in deg cm2 dmol1, using a mean residue weight of 110. Percentages of secondary structures were calculated by applying the Sreerama and Woody method (Sreerama and Woody, 1994). 2.6. Differential scanning calorimetry analysis Differential scanning calorimetry (DSC) analysis was carried on a Mettle 882 DSC (Mettle-Toledo, Switzerland). The samples for DSC measurements were scanned at a heating rate of 1 C /min from 20 to 90 C. The onset transition temperature (Tm) and calorimetric enthalpy (DH) were used as conformational stability indicating thermodynamic parameters. Increase in DH and Tm of the MTGase was interpreted as an indication of stabilizing effect provided by different polyols. All data manipulations were performed by using Origin software provided with the DSC. 2.7. Sodium dodecylsulfate–polyacrylamide gel electrophoresis The cross-linking reactions catalyzed by MTGase were carried out at 37 C in 20 mmol/L phosphate buffer (pH 6.5) containing 1% (w/v) casein as the substrate. The reaction mixtures were incubated for 10 min, then stopped by directly mixing with the sample buffer (2·) of electrophoresis and analyzed by SDS–PAGE according to the method of Laemmli (Laemmli, 1970). A 12.5% separating gel was used. The proteins were stained with a 0.1% solution of Coomasiie Brilliant Blue R-250.
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3. Results and discussion
100
3.1. Thermal stability of MTGase 80
Flourescence (%)
As was expected the thermal inactivation kinetics of the MTGase were fitted using one-step inactivation model according to Fig. 1. The inactivation rate of the enzyme was calculated by first-order expression: ln[Ut/U0] = kt. The k (inactivation rate constant or first-order rate constant) values were calculated from a plot of ln[Ut/U0] versus t at a particular temperature. The thermal inactivation of the enzyme at different temperature could be distinguished by the differences in the slopes determined by linear regression (Fig. 1). The transglutaminase produced by S. hygroscopicus was very stable below 40 C. The model parameters: inactivation rate constants (k, min1) and half-lives (t1/2, min) for MTGase at different temperatures (20 C, 30 C and 40 C) were 0.0009, 0.0016, 0.0072 and 770, 433, 96, respectively after calculation with the equations: ln[Ut/U0] = kt and t1/2 = ln2/k. When the temperature was above 50 C, the enzyme was inactivated rapidly and the inactivation rate constants (k, min1) and half-lives (t1/2, min) at 50 C and 60 C were 0.0350, 0.0828 and 20, 8, respectively.
40
0 min 20 min 40 min 60 min
20
0 300
20
30
Time (min) 60
100
-2
ln (Ut/U0)
-4
360
380
400
0.2
Absorbance
0.15
0.1
120
0
340
Fig. 2. Fluorescence emission spectra of MTGase after different periods of incubation at 60 C.
0.05
10
320
Wavelength (nm)
3.2. Conformational changes during heat treatment of the MTGase Changes in the fluorescence emission spectra of the MTGase after 0, 20, 40 and 60 min of incubation at 60 C were shown in Fig. 2. The fluorescence emission intensity markedly increased with prolonged heat treatment periods at 60 C and no obvious red or blue shift occurred to the fluorescence emission spectra. Fig. 3 showed the effect of heat treatment time on the ultraviolet spectra of the enzyme. The native MTGase exhibited an absorption peak at about 275 nm which was contributed
60
0 200
0 min 20 min 40 min 60 min 220
240
260
280
300
Wavelength (nm) Fig. 3. UV-absorbance spectra of MTGase after different periods of incubation at 60 C.
-6 -8 -10 -12
Fig. 1. Kinetics of thermal inactivation of MTGase from Streptomyces hygroscopicus. (4) 20 C: y = 0.0009x, R2 = 0.9886; (h) 30 C: y = 0.0016x, R2 = 0.9893; (s) 40 C: y = 0.0072x, R2 = 0.9937; (·) 50 C: y = 0.0350x, R2 = 0.9918; (–) 60 C: y = 0.0828x, R2 = 0.999.
by Tyr and Try residual in the protein molecule. The absorption significantly increased in magnitude after 20 min of incubation at 60 C. When the heat treatment time was prolonged further, the peak increased more slowly. No evident changes occurred of the absorption intensity for the enzyme treated at 60 C for 60 min by comparing with that for 40 min. This result was consistent with that of the fluorescence emission spectra. The spectral results indicated that the ordered structure was almost completely in an unfolded state after a 40-min incubation
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at 60 C and all the enzyme activity was lost simultaneously. Fig. 4 depicted the far-UV CD spectra of the MTGase from S. hygroscopicus when the temperature raised to 50 C and 60 C. The native MTGase from S. hygroscopicus was a a + b protein from the CD spectra with separate a-helix and b-sheet rich regions. The CD spectra of such proteins in water are dominated by the a-helix portion, exhibiting negative bands at around 208 and 222 nm and a positive band below 195 nm (Manavalan and Johnson, 1983). The MTGase contains about 20.5% a- helix and 33.1% b-sheet, which was quite similar to that from Streptoverticillium mobaraense (24.5% a-helix, 23.8% b-strand, 21.1% b-turn and 30.7% unordered structure) (Mene´ndez et al., 2006). With the temperature raised to 50 C, the intensities of the positive band and the negative CD bands decreased, without significant changes in the spectral form and marked wavelength shifts. The typical a-helix structure still retained and Sreerama and Woody method yielded 17.5% a-helix and 32.4% b-strand (Table 1). The unordered structure content increased from 46.6% to 50.1%. As the temperature increased further to 60 C, the intensity of the negative peaks at 208 nm and 220 nm decreased significantly and a pronounced blue shift at 208 nm was observed on the CD spectra. Analysis of the CD spectra
Fig. 4. Far-UV CD spectra of MTGase at different temperature.
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Table 1 Secondary structure and relative activity of MTGase at different temperatures Temperature (C)
a-Helix (%)
b-Sheet (%)
Turn + coil (%)
Relative activity (%)
25 50 60
20.5 17.5 12.5
33.1 32.4 28.9
46.6 50.1 58.5
100 60 20
of the denatured MTGase revealed that some of the secondary structure elements of this enzyme persisted even under such condition. The a-helix structure content of the enzyme decreased to 12.5% and the unordered structure content increased to 58.5% continually. Because it should take some time for the enzyme to unfold the natural structure, even at 60 C, 20% relative activity was still detected by using the activity determination method described above (using the activity of a sample without incubation as 100%). 3.3. Effect of polyols on thermal inactivation of MTGase Polyols have long been regarded as protein stabilizers (Hafedh et al., 2001). To examine the protective effect of some stabilizers on thermal inactivation of MTGase, the residual activity was determined after incubation at 50 C for 30 min in the presence of PEG, sorbitol, glycerol, sucrose, fucose and maltodextrin, respectively. As shown in Fig. 5, after a 30-min incubation at 50 C, about 70% activity of the enzyme was lost. At the given concentrations of different stabilizers, glycerol and sorbitol exhibited a higher stabilizing effect and about 75% activity of the enzyme was preserved. The nonpolar polyol PEG also played the same role as maltodextrin in protecting MTGase from heat denaturation at 50 C. The apparent halflives of the enzyme increased to 38 and 44 min approximately at 50 C by adding PEG and glycerol, respectively when compared to the control sample without any stabilizers (t1/2, 20 min) after calculation by using the first-order
Fig. 5. Effect of different stabilizers on the residual activity of MTGase after 30 min of incubation at 50 C with PEG at a concentration of 0.8 mol/L and with 10% (v/v) sorbitol, glycerol, sucrose, fucose and maltodextrin, respectively.
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expression: ln[Ut/U0] = kt (Fig. 6). Even the incubation temperature raised to 60 C, PEG and glycerol enhanced the thermal stability of the enzyme and the half-lives increased from 8 min to 16 and 21 min, respectively (Fig. 6). The improvement of thermal stability by adding polyols was probably due to the reinforcement of the hydrophobic interactions among nonpolar amino acids inside the enzyme molecules, and thus increased their resistance to inactivation, since it had been reported that polyhydroxy compounds modify the structure of water and/or strengthen hydrophobic interactions among nonpolar amino acids inside the protein molecules (Back et al., 1979). Moreover, PEG is a nonpolar polyol and can evidently increase the viscosity of the enzyme solution, which may cause reduction of chemical or biological reaction rate which could result in inactivation of the enzyme (Lee and Choo, 1989). 3.4. Analysis of protective effect of polyols by DSC and SDS–PAGE DSC is ideally suited to the study of protein thermal denaturation in solution since it measures the forces stabilizing the conformational structure directly and is therefore model independent (Richard et al., 1998). Thermograms obtained by DSC for MTGase without any additives presented one main peak. This peak was an endothermic one which occurred at a temperature of 60.166 C which was defined as the onset transition temperature (Tm) of the folding/unfolding transition to the MTGase. The DH value for the transition was 4.5008 ± 0.02 J/g (Table 2). No peaks were visible on the rescan, which means that all tranTime (min) 30
60
90
120
0
-1
ln(Ut/U0)
-2
-3
-4
-5
-6
Fig. 6. Kinetics of thermal inactivation of MTGase from Streptomyces hygroscopicus in the presence of glycerol and PEG at 50 C (—) and 60 C (—). (d): control; (4): glycerol; (h): PEG.
Table 2 Effect of different stabilizers on onset transition temperature and calorimetric enthalpy of MTGase Polyols
Transition temperature, Tm (C)
Calorimetric enthalpy, DH (J/ g)
Control Sucrose Sorbitol Glycerol
60.166 ± 0.3 66.257 ± 0.2 64.981 ± 0.3 68.346 ± 0.6
4.5008 ± 0.02 4.5760 ± 0.02 4.7003 ± 0.03 4.8001 ± 0.05
sitions were irreversible after heating to 90 C. The effects of polyols on the conformational stability of the enzyme against heat treatment were deduced from the shift of this transition peak and the values for Tm and DH were shown in Table 2. It was clear that all of the polyols examined caused a higher Tm and a significant increase in DH value in comparison with the control. The Tm and DH values of the MTGase containing glycerol increased to 68.346 ± 0.6 C and 4.8001 ± 0.05 J/g, which indicated that glycerol exhibited a higher stabilizing effect. The presence of sucrose and sorbitol also enhanced the Tm and DH values of the enzyme to 66.257 ± 0.2 C, 64.981 ± 0.3 C and 4.5760 ± 0.02 J/g, 4.7003 ± 0.03 J/g, respectively. In addition, the DSC thermograms of the MTGase in the absence (control) and presence of the above three stabilizers were taken after 50 min of incubation at 60 C and chill immediately on ice (the thermograms not shown here). The MTGase without any additives did not show any transition peaks, which might be because of irreversible thermal denaturation of the enzyme. The enzyme mixed with polyols still showed a distinct transition peak with a significantly greater Tm values in comparison with the control. The results indicated that the polyols maintained the ordered structure to some degree during thermal unfolding of the enzyme. Glycerol was taken as an example for estimate of the protective effect of the polyols on MTGase against thermal inactivation by SDS–PAGE analysis. The enzyme used was pre-incubated at 60 C for different periods, aliquots were then withdrawn and chilled immediately. A casein-based system was used and the SDS-gel electrophoretic patterns obtained after cross-linking with MTGase. On the SDS– PAGE pattern, new protein biopolymers cross-linked by transglutaminase can congregate at the interface of the stacking and separating gel or stop at the upper part of the separating gel due to the increase of molecular weight. The cross-linking reaction conditions of all the samples: the reaction temperature, reaction time, pH, protein concentration and the enzyme amount were the same except for the enzyme containing glycerol in samples d, e, f and without glycerol in samples a, b, c. (Fig. 7). Lane a indicated the crosslinked products catalyzed by MTGase not containing glycerol after incubation for 10 min. There were some biopolymers at the interface of the stacking and separating gel and more new high molecular weight proteins appeared at the upper part of the separating gel by comparing with lane o, which suggested that
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Fig. 7. SDS–PAGE analysis of cross-linked casein at 37 C for 10 min by MTGase after different periods of incubation at 60 C. (s: casein solution without MTG; a, b, c: casein solution with MTG after incubation of 10 min. 20 min, 30 min at 60 C ; d, e, f: casein solution with MTGase containing 10% glycerol after incubation of 10 min. 20 min, 30 min at 60 C).
cross-linking reaction occurred because of the residual enzyme activity. Lane d showed the cross-linked products catalyzed by MTGase with 10% glycerol after incubation for 10 min. By comparing with lane a, there were much more biopolymers on the top of separating and stacking gel and the amount of low molecular weight proteins decreased at the same time. The results indicated that more enzyme activity was retained by adding glycerol and enhanced the cross-linking reaction efficiency. Lane e showed the products catalyzed by MTGase containing glycerol after 20 min of incubation. The amount of the biopolymers induced by MTGase was much less than that in lanes a and d, which suggested that the majority of the enzyme activity was lost after incubation for 20 min despite the addition of glycerol. In the absence of glycerol, the extent of crosslinking of casein catalyzed by the MTGase was remarkably reduced as a function of pre-incubation time of the enzyme, and nearly completely inactivated after incubation for 30 min at 60 C (lane c). However, cross-linking of casein was considerably improved by adding glycerol during the pre-incubation (lanes d, e and f) and more new high MW biopolymers increased on the top of separating and stacking gel by comparing with the MTGase without glycerol. The results showed that glycerol was involved in the stabilization of MTGase by reinforcing the hydrophobic interactions among nonpolar amino acids inside the protein molecules, and thus rendering them resistant to thermal inactivation mentioned above (Back et al., 1979). 4. Conclusions TGase has captured peoples’ intense interest due to its attractive potential application for its special catalytic character. It is very important to obtain the enzyme with high thermal stability and activity for its preparation and application in industrial purposes. Considering this fact, we report here the thermal stability and conformational
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changes of transglutaminase produced by a newly isolated strain S. hygroscopicus. The enzyme was stable under 40 C. Some polyols: sorbitol, sucrose, fucose, maltodextrin, polyethylene glycol 6000 (PEG) and glycerol, could improve the thermal stability of the enzyme. The half-lives for the MTGase at 50 C and 60 C were increased from 20 min and 8 min to 40 min and 20 min, respectively by mixing with 10% glycerol, Spectroscopic studies of the MTGase indicated conformational transition from ordered secondary structural elements (a/b protein) to unordered structures during thermal treatment. DSC and SDS–PAGE analysis of the cross-linked products by MTGase confirmed the protective effect of polyols on the enzyme against thermal denaturation. The MTGase mixed with these polyols showed a distinct transition peak with a significant increase in Tm and DH values by comparing with the control. From the results above, the possibility of stabilizing the MTGase against thermal inactivation in the presence of polyols would be of great interest for industrial preparation and storage, which is important to make this enzyme a good candidate for application in different industries. Acknowledgements This project was financially supported by the Open Project Program of Key Laboratory of Eco-Textiles (Southern Yangtze University, China, No. KLET0623), program for New Century Excellent Talents in University and a grant from National Science Fund for Distinguished Young Scholars. References Aeschlimann, D., Paulsson, M., 1994. Transglutaminase: protein crosslinking enzymes in tissues and body fluids. Thromb. Haemostasis 71, 402–415. Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H., Motoki, M., 1989. Purification and characterization of a novel transglutaminase derived from micro-organisms. Agric. Biol. Chem. 53, 2613–2617. Back, J.F., Oakenfull, D., Smith, M.B., 1979. Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry 18, 5191–5196. Barros, S.L.H., Assmann, F., Zachia, A.M.A., 2003. Purification and Properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment. Biotechnol. Appl. Biochem. 37, 295–299. Cortez, J., Bonner, P.L.R., Griffin, M., 2004. Application of transglutaminase in the modification of wool textile. Enzyme Microb. Technol. 34, 64–72. Cowan, D.A., 1997. Thermophilic proteins: stability and function in aqueous and organic solvents. Comp. Biochem. Physiol. A Physiol 118, 429–438. Cui, L., Zhang, D., Huang, L., Liu, H., Du, G., Chen, J., 2006. Stabilization of a new microbial transglutaminase from Streptomyces hygroscopicus WSH03 – 13 by spray drying. Process Biochem. 41, 1427–1431. Cui, L., Du, G., Zhang, D., Liu, H., Chen, J., 2007. Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus. Food Chem., doi:10.1016/j.foodchem.2007.04.020.
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