- Email: [email protected]
Enhanced activity of immobilized transglutaminase for cleaner production technologies
Journal of Cleaner Production 240 (2019) 118218
Contents lists available at ScienceDirect
Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Enhanced activity of immobilized transglutaminase for cleaner production technologies Marjetka Gajsek a, b, Urska Jan ci c a, Katja Vasi c a, Zeljko Knez a, c, Maja Leitgeb a, c, * a Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova ulica 17, SI2000 Maribor, Slovenia b Kozmetika Afrodita d.o.o., Kidriceva 54, SI-3250, Rogaska Slatina, Slovenia c Faculty of Medicine, University of Maribor, Smetanova 17, SI-2000, Maribor, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 April 2019 Received in revised form 26 August 2019 Accepted 28 August 2019 Available online 3 September 2019
The enzyme transglutaminase (TGM) has a very important role in applications in various industries. Potentially, a substrate for microbial TGM may be any proteinaceous molecule with glutamine and lysine residues, which broadens the importance of microbial TGM for clean industrial applications or for the use of biosensors. Because stabilization and reusability of an enzyme is an economically important goal for enzymatic processes, the purpose of this study was immobilization of TGM onto magnetic nanoparticles (MNPs) synthesized by the co-precipitation of Fe2þ in Fe3þ ions and modified with carboxymethyldextran (CMD). Synthesized nanoparticles were firstly activated with a cross-linking agent (glutaraldehyde (GA) and/or pentaethylenehexamine (PEHA)) and then used for covalent immobilization of the enzyme. Activity of immobilized TGM was determined and compared with activity of the free enzyme. Under optimal immobilization conditions, the enzyme was hyperactivated and showed 99% immobilization efficiency and 110% residual activity. The immobilized TGM showed excellent thermal stability at 50 C and 70 C in comparison with non-immobilized enzyme and very good reusability and can be used for cleaner production in different real life industrial applications, such as in leather, textile and wool industry. © 2019 Elsevier Ltd. All rights reserved.
Handling editor: Prof. Jiri Jaromir Klemes Keywords: Immobilization Magnetic nanoparticles Transglutaminase Enzyme stabilization Cleaner technologies
1. Introduction Transglutaminases (TGM, EC 2.3.2.13) are enzymes that have attracted wide interest from both scientific and applied points of view (Mehta et al., 1997). They belong to the class of transferases and catalyze an acyl transfer reaction between the g-carboxamide groups of glutamine residue (acyl donor) and a variety of primary amines (acyl acceptors), including the ε-amino group of lysine residues (Motoki and Seguro, 1998). This enzyme polymerizes proteins intra- or intermolecularly and forms bonds with high resistance to proteolytic degradation (Yang et al., 2011). It is a promising biocatalytic alternative to classical organic chemistry for amide bond synthesis (Rachel and Pelletier, 2013; Zhang et al., 2010). TGMs are involved in many physiological functions, such as blood coagulation, wound healing, epidermal keratinization, and
* Corresponding author. Faculty of Medicine, University of Maribor, Smetanova 17, SI-2000, Maribor, Slovenia. E-mail address: [email protected] (M. Leitgeb). https://doi.org/10.1016/j.jclepro.2019.118218 0959-6526/© 2019 Elsevier Ltd. All rights reserved.
stiffening of the erythrocyte membrane (Motoki and Seguro, 1998). TGMs also play a role in stabilization of photosynthetic complexes in the chloroplast, modification of cytoskeletal proteins, abiotic and biotic stresses, aging, and programmed cell death in plants. Currently, they are used extensively in the food industry to affect the structure of protein systems and cause changes leading to improved texture, emulsifying properties, thermal stability, gelation, syneresis and increased water-binding capacity (Gaspar and es-Favoni, 2015). In other sectors, the potential applications de Go of TGM include the building of collagen- or gelatin-based scaffolds for the construction of bioartificial organs, site-specific protein conjugation with DNA in biotechnology and materials science, textile and leather processing (Yang et al., 2011). The enzyme is very unstable if it is dissolved in water, however, and its inactivation is influenced by heating, strong acids and bases, and some organic solvents. All these adverse conditions greatly limit the application of the enzyme (Na et al., 2012). Such limitations could be eliminated by using TGM in an immobilized form. The immobilized enzyme could be easily separated from the solution, could function in a much wider pH and temperature range, should have higher
2
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
thermal stability compared to the free enzyme, and it could also be reused. However, immobilization can reduce the catalytic activity of the enzyme, so optimization of the immobilization method is crucial in the preparation of an efficient and economical biocatalyst (Zhou et al., 2016). TGM has been widely used in the textile, wool and leather industries, since it can be produced at an industrial level easily by simple fermentation (Tesfaw and Assefa, 2014). In the wool industry, TGM repairs the damage caused by treatments in which an array of chemicals and enzymes is employed for scouring, carding, gilling, combing, drafting, spinning and twisting, which are processing steps for wearable clothes (Cortez et al., 2005; Lenting et al., 2006). TGM treatment followed by hydrogen peroxide, protease and ultrasonification shows that TGM can improve the crease resistance of silk fabric and enhance tensile breaking strength caused by pretreatments (Zhu et al., 2013). In the leather industry, filling is one of the essential steps in processing. The most commonly used natural products in these steps are glucose, flour and gum; however, gelatin and casein are also used as effective filling materials. Treatment of casein with TGM results in highly polymerized gelatines which are able to fill the leather. Therefore, with TGM treatment, expensive filling treatment of leather materials could be replaced with inexpensive proteinaceous industrial byproducts (Taylor et al., 2007; Tesfaw and Assefa, 2014; Zhu and Tramper, 2008). Since the leather industry produces many pollutants that are toxic (Chaudhary and Pati, 2016; Ramamurthy et al., 2014; Silambarasan et al., 2015) and are discharged directly into the soil, causing soil pollution, they affect human health through water and food. Immobilized TGM treatment provides “green”, but most importantly environmentally friendly approaches to clean leather and textile production. TGM is also widely known to modify food proteins, and it is a promising tool to improve functional properties of protein-based products, such as gelation properties, solubility, emulsifying capacity, foaming, viscosity and elasticity. Possibilities for TGM application can be found in many dairy products such as milk proteins, cheese products, fermented milks, milk powders, and caseinate products (Romeih and Walker, 2017). Functional properties of foods have been improved using TGM in this way. TGM has also been used in the process of enhancing dough-making rheological performance of wheat flour via vital gluten (VG) supplementation. For this purpose, 4 different levels of TGM were used in the experimental design, where TGM and VG changed dough rheological behaviour, which offers opportunities for bread quality improvement (Bardini et al., 2018). Tissue TGM obtained from guinea pig liver was also used to develop a hepatocyte-embedded hydrogel-filled macroporous scaffold culture technology to establish a fundamental technology for liver tissue technology (Ijima et al., 2010). Microbial TGM was used in the process of biotinylation of enhanced green fluorescent protein and bacterial alkaline phosphatase in order to prepare streptavidin-immobilized hydrogel (Moriyama et al., 2013). With the rapid development of nanotechnology, nano-carriers (nanoparticles, nanofibers, nanotubes, nanosheets) are becoming increasingly attractive for immobilization of enzymes. Among them, MNPs have attracted a great deal of attention because of their unique properties (superparamagnetism, high surface area, large surface-to-volume ratio). Magnetic metal nanoparticles are also used to immobilize enzymes because of the low cost of synthesis and negligible toxicity (Xu et al., 2014; Yang et al., 2016). It has also been found that the stability and activity of enzymes increase if they are immobilized on such materials (Ahmad and Sardar, 2015). However, uncoated iron oxide nanoparticles are generally unstable in strong acidic solutions and undergo leaching (Xu et al., 2014). They also tend to agglomerate as a result of strong magnetic attraction among particles, van der Waals forces and high energy
surface. This could be avoided by surface modification of nanoparticles via loading of other target chemicals or biological materials during or after the synthesis process. Various materials have been used as coating materials in aqueous solutions, for example, oleic acid, PEG, PVA, dextran, starch and chitosan (Ali et al., 2016; Hong et al., 2008; Xu et al., 2014). Among them, carboxymethyl dextran (CMD) has outstanding properties - it is biodegradable, biocompatible and bioactive (Ferk et al., 2012). Compared to pure dextran, CMD has additional carboxyl groups which can be used for the covalent bonding of additional molecules (Makovec et al., 2015). Some studies on TGM immobilization already exist. This enzyme was immobilized onto a polyethersulfonate membrane surface, where enzymatic membrane filtration efficiency and a mechanism for catalysis of protein cross-linking and separation from cheese whey were investigated. In this case, TGM was covalently immobilized and a protein recovery rate of 85% in a TGM enzymatic membrane reactor (EMR) was achieved (Wang et al., 2018). Another study describes overcoming mass transfer limitations of immobilized TGM, where TGM was covalently immobilized on a thermo-responsive carboxylated poly(N-isopropylacrylamide) (pNIPAM). After conjugation to pNIPAM, TGM showed a down shifting optimal temperature as well as better thermo-stability. The pNIPAM-TGM conjugate was sensitive to change in temperature with a low critical solution temperature (LCST) at around 39 C, leading to easy separation of the biocatalyst after the catalysis reaction (Zhou et al., 2016). An easy approach for the production of reusable immobilized recombinant Escherichia coli biotin ligase (BirA) onto amine-modified magnetic microspheres (MMS) via covalent cross-linking was proposed using microbial TGM. The site-specifically immobilized BirA exhibited approximately 95% of the enzymatic activity of the free BirA, without a significant loss in intrinsic activity after 10 rounds of recycling (P > 0.05) (Yu et al., 2016). TGM antigen was also immobilized on polyamidoamine (PAMAM) to form a nanoprobe for sensing specific anti-tissue transglutaminase (anti-TGM) antibodies (immunoglobin A isotype) in human serum. The sensor was highly specific to antitissue transglutaminase antibodies and showed negligible response to non-specific serum proteins. Using cross-linkers, such as GA and PEHA for functionalization of MNPs was widely used, but not in a relation to immobilizing TGM. As there are not many studies based on immobilizing TGM onto magnetic supports, the goal of our study was to optimize immobilization of the TGM onto a magnetic nano-carrier, modified with CMD, which has not yet been widely investigated. The covalent attachment of TGM to the support surface via GA and PEHA functionalization was expected to result in a greater stability of the immobilized TGM. Our goal was to prepare an economical biocatalyst that is non-toxic and biodegradable with high activity, improved stability and reusability, which would reduce the cost of enzyme biocatalysis for industrial applications. Such immobilized TGM would have a possibility to be used for cleaner production in different real life industrial applications, such as in leather, textile and wool industry. First, MNPs were prepared, and in their modified and activated form (using GA and PEHA functionalization), they were used for immobilization of TGM (Probind TXo). Immobilization efficiency and residual activity of immobilized TGM was determined, and thermal stability of immobilized and free TGM and reusability of the immobilized TGM were investigated. 2. Materials and methods 2.1. Reagents and materials Transglutaminase, Probind TXo was obtained from BDF Natural Ingredients, with initial activity of 0.07 U/mL and initial protein
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
concentration of 0.46 mg/mL. Carboxymethyl-dextran (CMD) sodium salt, bovine serum albumin (BSA), egg albumin (EA), glutaraldehyde (GA), pentaethylenehexamine (PEHA), ethanol (95%), LGlutamic acid g-monohydroxamate, L-glutathione reduced, hydroxylamine hydrochloride (99%), trichloroacetic acid and Tris buffer (Trizma base) were purchased from Sigma-Aldrich. Z-Glutaminylglycine (Z-Gln-Gly-OH) was purchased from Zedira GmbH, Germany. Iron (II) chloride tetrahydrate, iron (III) chloride hexahydrate, Coomassie brilliant blue G-250, phosphoric acid (85%), hydrochloric acid (37%) and acetic acid (100%) were purchased from Merck. Ammonia was purchased from Chem-Lab and calcium chloride from Kemika. 2.1.1. Preparation of MNPs modified with CMD MNPs were prepared using a co-precipitation method. In the procedure, Fe2þ (0.101 M) and Fe3þ (0.204 M) ions were dispersed in milliQ water under nitrogen flow with vigorous stirring and the temperature was raised to 85 C. Then, CMD-ammonia solution (5 g dextran per 20 mL H2O and 5 mL 25% NH3) was added in several successive small additions over a period of 20 min. The suspension was aged at 85 C for 1 h and the colour of the suspension changed from brown to black. The cooled suspension was then magnetically decanted and washed several times with milliQ water. Then, prepared surface-modified CMD coated nanoparticles (CMD-MNPs) were dried at 50 C and used for immobilization. 2.1.2. Preparation of CMD-MNPs, modified with citric acid Following the co-precipitation method of Fe2þ and Fe3þ ions and the addition of CMD-ammonia solution (5 g dextran per 20 mL H2O and 5 mL 25% NH3), as described in section 2.2, the solution was vigorously stirred at 85 C for 10 min. After 10 min, 2.5 mL of citric acid (0.5 g/mL) was added dropwise and the solution was vigorously stirred for 90 min at 90 C under nitrogen flow. The cooled suspension was then magnetically decanted and washed several times with milliQ water. The prepared MNPs modified with CMD and citric acid (CMD-citric) were dried at 50 C. 2.1.3. Preparation of CMD-MNPs modified with oleic acid Following the co-precipitation method of Fe2þ and Fe3þ ions and addition of CMD-ammonia solution (5 g dextran per 20 mL H2O and 5 mL 25% NH3), as described in section 2.2, the solution was vigorously stirred at 85 C for 10 min. After 10 min, 3 mL of oleic acid was added dropwise and the solution was vigorously stirred for another 90 min at 90 C under nitrogen flow. The cooled suspension was then magnetically decanted and washed several times with milliQ water. The prepared MNPs, modified with CMD and oleic acid (CMD-oleic), were dried at 50 C. 2.1.4. Immobilization of TGM onto nanoparticles The immobilization was carried out using a GA and/or PEHA chemical activation procedure. Synthesized nanoparticles were stirred in a solution of GA and/or PEHA in a Tris buffer with pH 6. The mixture was shaken at 400 rpm for 2 h at room temperature. The activated nanoparticles were then separated from the supernatant in the presence of a magnetic field and further used for immobilization of TGM. TGM was added to activated MNPs, which were previous dispersed in a Tris buffer with pH 6 and the reaction mixture was shaken for 4 h at 10 C. After immobilization of TGM onto CMD nanoparticles, the unbound enzyme was removed by washing with a Tris buffer with pH 6. Supernatants were separated from nanoparticles using a magnet and further used to determine the concentration of the unbound enzyme and thus the immobilization efficiency. Activity of immobilized TGM was also determined.
3
2.1.5. Protein concentration Protein concentration was quantified with Bradford microcrystalline assay (Bradford, 1976), using BSA as calibration standard. 20 mL of enzyme and 1 mL of Bradford reagent were mixed and measured at 595 nm on UV/VIS spectrophotometer after 15 min incubation at room temperature. Control sample contained equivalent volume of Milli-Q water instead of enzyme solution. Immobilization efficiency was calculated by Equation (1):
Immobilization efficiencyð%Þ ¼
ðci cs Þ *100 ci
(1)
where: ci - concentration of TGM. cs - concentration of unbound TGM, collected in supernatant. Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.
2.1.6. Activity assay of TGM TGM activity was determined by a colorimetric hydroxamate procedure (Fig. 1) in which Z-Gln-Gly was used as the amine acceptor substrate and hydroxylamine as amine donor. In the presence of TGM, hydoxylamine was incorporated into Z-Gln-Gly to form Z-glutamyl-hydroxamate-glycine, which developed a coloured complex with iron (III) detectable at 525 nm. One unit of TGM activity was defined as the amount of enzyme that causes the formation of 1.0 mmole of hydroxamate per minute by catalyzing the reaction between Z-Gln-Gly and hydroxylamine at pH 6.0 at 37 C. L-glutamic acid g-monohydroxamate was used as a standard probe. Activity (U/mL) of the enzyme was calculated according to Equation (2):
Activity
U mL
¼
Atest ð525nmÞ df ðEnM1 Þ ð10Þ ð1; 23Þ
(2)
EnM1 ¼ Atest ð525nmÞ 1:1 where: Atest (525 nm) - Absorbance of sample measured at 525 nm. df - dilution factor. 1.1-volume of standard (in mL). 1.23-volume of colour mix (in mL).
Fig. 1. Calorimetric assay for detection of TGM activity (Makovec et al., 2015).
4
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
10 - time of reaction (in min). Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%. 2.1.7. Thermal stability of free and immobilized TGM To study the thermal stability of free and immobilized TGM, it was exposed to two different temperatures for a defined time in 1 M Tris buffer adjusted to pH 6 and also in nonaqueous pure form without buffer. Temperatures chosen for thermal stability study of TGM were 50 C and 70 C, respectively. Incubation time at a certain temperature was 2 h, 4 h and 6 h. After incubation, the samples were brought to room temperature, substrates were added, and the residual activity of the enzyme was determined as described in section 2.4. The residual activity of TGM was calculated according to the enzyme activity before incubation at different temperatures. 2.1.8. Reusability assay The possibility of immobilized TGM reuse was evaluated through 6 consecutive reaction cycles. In the presence of TGM, hydoxylamine was incorporated into Z-Gln-Gly to form Z-glutamylhydroxamate-glycine. Activity of the immobilized TGM was determined after each reaction cycle by activity test for TGM. At the end of each reaction cycle, immobilized TGM was magnetically separated and washed with 1 M Tris buffer with pH 6 before being reused. The activity of the first cycle was defined as 100% activity of the immobilized TGM. Residual activity (%) was defined as the ratio between the enzyme activity at the end of each cycle and the enzyme activity from the first cycle. 3. Results and discussion 3.1. Optimization of TGM immobilization conditions onto MNPs The success of the immobilization procedure depends particularly upon the choice of the enzyme, the support and the immobilization method (Netto et al., 2013). In order to effectively immobilize TGM and improve enzyme stability, activity and reusability, suitable immobilization conditions are critical. Therefore, five factors affecting immobilization of TGM covalently bound onto MNPs were investigated.
Fig. 2. Effect of shaking speed on the immobilization efficiency and residual activity of TGM. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
feeders and the sequence of their addition were investigated in order to further optimize immobilization and maintain the highest residual TGM activity. It is well known that BSA facilitates enzyme immobilization when high concentrations of glutaraldehyde are detrimental to enzyme activity or when the enzyme has a low content of lysine residues. The carrier was activated either with PEHA, GA or combination of both. Albumins were chosen because they have proven to be effective enzyme stabilizers in some cases. They can stabilize the active enzyme structure by forming new bonds (hydrogen, electrostatic or donor-acceptor) between the enzyme and a carrier matric (Torchilin, 1991). BSA or EA with concentration 50 mg/mL was added at the same time as the enzyme (Fig. 3) and also separately so that at first, stabilizing protein was added and later the enzyme solution (Fig. 4). Other parameters were fixed (1 vol % GA or 10 vol % PEHA or combination of 10% PEHA þ0.25% GA, cross-linking time 2 h, immobilization time 4 h at 10 C). As shown in Figs. 3 and 4, the amount of immobilized enzyme significantly increased with the use of stabilizing proteins. When stabilizing proteins BSA or EA were used
3.1.1. Effect of shaking speed To determine the influence of shaking speed on immobilization efficiency and residual activity of TGM, experiments were performed under the following conditions: 2-h activation of the carrier with 10 vol % PEHA, 4-h immobilization of the mixture 100 mg/mL of TGM and 50 mg/mL of BSA at 10 C, and different shaking speeds (300, 400 or 500 rpm). As shown in Fig. 2, shaking speed does not significantly affect the TGM immobilization efficiency, but this cannot be claimed for residual activity of the enzyme, since it was highest (47%) at 400 rpm and lowest (20%) at 500 rpm. The obtained results confirm the importance of this parameter optimization when an immobilization procedure is introduced for a certain enzyme. It is assumed that at a higher shaking speed (500 rpm), TGM inactivation occurs due to strong shear forces. Inactivation is the result of destabilization of electrostatic, hydrophobic and hydrogen bonds of the enzyme, which leads to irreversible enzymatic denaturation and, consequently, lower residual TGM activity. Therefore, a shaking speed of 400 rpm was selected as optimal and all further experiments were done at this shaking speed. 3.1.2. Effect of stabilizing proteins addition The effect of different albumin proteins (BSA or EA) as proteic
Fig. 3. Effect of stabilizing proteins (BSA or EA): stabilizing protein was added together with the enzyme. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%).
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
together with TGM, immobilization efficiency reached 92% when BSA was used and 87% when EA was used (Fig. 3). When stabilizing proteins BSA or EA were added before the addition of the enzyme, immobilization efficiency was much higher (Fig. 4), and reached 98% when BSA was used and 99% when EA was used. Without BSA or EA, it was only 60% when nano-carrier was activated with a combination of 10 vol % PEHA and 0.25% GA. Moreover, the sequence of stabilizing protein addition is an important parameter that may influence enzyme activity. Indeed, in all cases the activity of immobilized TGM was higher when stabilizing protein was added before the addition of enzyme. From the results, it is clear that using only BSA as proteic feeder increases the activity of immobilized TGM. BSA, which contains 66 lysines in its sequence, increased the content of lysine residues, and the consequent crosslinking efficiency. By adding stabilizing protein BSA before the addition of the enzyme and using 10 vol % PEHA for activation of CMD-carrier, TGM was hyperactivated with a residual activity of 109%. Consequently, these parameters were selected as optimal and used for further optimization. 3.1.3. Effect of the TGM concentration To determine the effect of TGM concentration on immobilization efficiency and residual activity of the enzyme, TGM solutions with different concentrations (75, 100 and 150 mg/mL) were used. Other parameters were fixed (10% PEHA or combination of 10% PEHA þ 0.25% GA, 50 mg/mL BSA, cross-linking time 2 h, immobilization time 4 h at 10 C). As can be seen from Fig. 5, immobilization efficiency and residual activity of the enzyme strongly depend on the concentration of the enzyme. The amount of immobilized enzyme and residual activity increased with the increase of TGM concentration, but only up to 100 mg/mL of TGM. With further increase of TGM concentration, immobilization efficiency decreased. We assume that the enzyme immobilization had already reached its maximum at 100 mg/mL of TGM and the surface of the nano-carrier was saturated with the enzyme. The decrease in activity at high TGM concentration might also be related to the
5
aggregation of the enzyme, which makes some active sites of the enzyme hidden (Cortez et al., 2005). The same effect was observed by Eldin et al. (2015), where high beta-galactosidase concentration led consequently to increase the density of the immobilized enzyme molecules on the particles’ surface which in turn led to the rise of “protein-protein” interaction and a consequent reduction of catalytic activity. Although the presence of GA increases immobilization efficiency at low TGM concentration (75 mg/mL), it significantly reduces the enzyme activity at high TGM concentration (150 mg/ml). This effect comes even more to the point at optimal TGM concentration (100 mg/mL), where residual TGM activity of 110% in the case of using PEHA decreased to 70% in the case when GA was added in combination with PEHA. 3.1.4. Effect of immobilization temperature Another important factor influencing activity of the enzyme is immobilization temperature. Enzyme was immobilized onto a CMD-carrier at room temperature (without cooling) and at 10 C (with cooling). From Fig. 6, it may be observed that immobilization temperature does not influence immobilization efficiency, which is the same with or without cooling. But residual activity of TGM was remarkably higher when the enzyme was immobilized onto a carrier at 10 C. It was 110% when enzyme was immobilized onto CMD with cooling and carrier was activated with 10 vol % PEHA, and only 51% under the same conditions but for immobilization at room temperature. When EA was used as a stabilizing protein and the nano-carrier was activated with 10 vol % PEHA in combination with 0.25 vol % GA, the differences in residual TGM activity were lower e only 19%, but as in the previous case, maximal residual activity was lower as well (70% and 66%). 3.1.5. Effect of nano-carrier TGM was immobilized onto different nano-carriers, CMD-MNPs, CMD-oleic, CMD-citric and aminosilane MNPs, to study the effect of the cover layer of MNPs on TGM activity after immobilization.
Fig. 4. Effect of stabilizing proteins (BSA or EA): first stabilizing protein was added before the addition of enzyme. (Each experiment was performed triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
6
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
Fig. 5. Effect of TGM concentration on the immobilization efficiency and residual activity of immobilized TGM. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Fig. 6. Immobilization efficiency and residual activity of TGM immobilized onto CMD nano-carrier with or without cooling. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Comparison of the residual TGM activity of such prepared biocomposites is shown in Fig. 7. Immobilization of TGM was carried out for 4 h at 10 C on a shaker at 400 rpm and a concentration of the enzyme 100 mg/mL, concentration of BSA or EA 50 mg/mL and 10 vol % PEHA or a combination of 10 vol % PEHA and 0.25 vol % GA. The nano-carriers were first activated with PEHA or the combination of PEHA and GA, then a stabilizing protein (BSA or EA) was
added, and finally TGM was immobilized. The results showed that with the selected parameters, the highest residual activity of the enzyme was achieved with CMD-MNPs (110%) and activation of the nano-carrier with 10 vol % PEHA. Residual activity of TGM immobilized onto CMD-oleic, activated with a combination of 10 vol % PEHA and 0.25 vol % GA, was 103%. The lowest residual activity (22%) was shown by TGM, immobilized on CMD-citric, activated
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
7
Fig. 7. Residual activity of TGM immobilized onto different nano-carriers: CMD-MNPs, CMD-oleic, CMD-citric and aminosilane MNPs. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
with a combination of 10 vol % PEHA and 0.25 vol % GA. For CMDMNPs, CMD-oleic and CMD-citric, the use of the BSA stabilizing protein was more appropriate than the use of EA. Conversely, for aminosilane MNPs, residual activity was higher when using EA protein. The choice of carrier and immobilization conditions therefore has a significant effect on residual TGM activity. 3.1.6. Stability of immobilized TGM Evolution of any new immobilization protocols focuses primarily on percentage enzyme recovery and operational stability. Therefore, the thermal stability of immobilized and nonimmobilized TGM and reusability of immobilized TGM were also studied. 3.1.7. Thermal stability of immobilized TGM Thermal stability of the enzyme is a very important parameter for its industrial applications, because it determines the limits for use and reuse of the enzyme, and therefore process costs (Shi et al., 2011). There is a shift in the optimum temperature of immobilized enzymes towards higher temperatures. During enzyme immobilization, free movement of enzyme molecules is obstructed, even at higher temperatures. Thus, enzyme denaturation is not observed due to protection of amino acids at the active site as well as on the surface (Dwevedi, 2016). In general, covalently immobilized enzymes are more resistant to heat than those in the soluble form. Therefore, thermal stability of TGM immobilized on two different nano-carriers, CMD-MNPs and CMD-oleic, both prepared under optimal conditions (CMD-MNPs: 10 vol % PEHA, 50 mg/mL BSA, 100 mg/mL TGM, CMD-oleic: 10 vol % PEHA þ0.25 vol % GA, 50 mg/ mL BSA, 100 mg/mL TGM, 4-h immobilization of the enzyme at 10 C) was tested and compared with that of pure, nonimmobilized enzyme. TGM was incubated at 50 C and 70 C in Tris buffer, adjusted to pH 6 and, in the pure form, in a non-aqueous medium. As shown in Fig. 8 and Fig. 9, immobilized TGM was less sensitive to temperature change than the free enzyme, which almost completely denaturated after 2 h of incubation at both
temperatures. This confirms better thermal stability of immobilized TGM compared to the free enzyme. At 50 C (Fig. 8), enzyme activity decreases as the time of incubation increases from 2 to 6 h in all cases. The enzyme, immobilized on both carriers, CMD-MNPs and CMD-oleic, maintained almost 40% of its activity after 6-h incubation in the buffer. Residual activity of the immobilized enzyme was even higher when TGM was incubated in a non-aqueous medium. This was expected, since it is well known that water is needed for enzyme molecular changes to reach inactivation. The TGMs’ residual activity in the non-aqueous medium was approximately 60% for CMD-oleic, and 66% for CMD-MNPs. The nonimmobilized enzyme was almost completely inactive after 2-h incubation at 50 C. The results show that the stability of TGM depends on its shape or state. Immobilization results in strengthening of the enzyme structure and provides a protective effect against heat denaturation (Shi et al., 2011). Consequently, immobilized enzyme could work in tougher environments with minimal activity loss. Better stability of the immobilized TGM can also be associated with a multi-point covalent bond between the TGM and the carrier, which prevents changes in enzyme structure at high temperatures, leading to its deactivation. The obtained results are promising for applications of such immobilized TGMs in industrial use. In other words, after incubation at 50 C, the very high productivity of non-immobilized TGM, which was 111.88 mg/(min mg enz), drastically decreased after 6 h of incubation to 3.78 mg/(min mgenz), which is only 3.4% of the initial value. As can be observed in Table 1, initial productivity for CMD-MNPs and CMD-oleic (incubated at 50 C in a buffer or in a non-aqueous medium) was higher than that of the non-immobilized enzyme. We would expect it to be the other way around as a result of diffusion limitations when using immobilized enzyme. Another advantage is that the productivity decrease after 6 h incubation at 50 C for CMD-MNPs and CMDoleic is, compared to non-immobilized TGM, much lower. The 6 h incubation in a non-aqueous medium resulted in a productivity drop to 97.25 mg/(min mgenz) for CMD-MNPs, which is only 64% of the initial productivity.
8
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
Fig. 8. Thermal stability of free and immobilized TGM at 50 C. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Fig. 9. Thermal stability of free and immobilized TGM at 70 C. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Since it is well known that structural changes that cause inactivation of the enzyme molecule at high temperatures are more intensive in an aqueous medium, the influence of immobilized TGM incubation at elevated temperature on the productivity of the enzyme was studied. The results show that even in an aqueous medium where a significant decrease in the productivity after 6 h incubation at 50 C is expected, this was 54.44 mg/(min mgenz) for CMD-MNPs, which is much higher that of the non-immobilized enzyme. This confirms excellent stabilization of the
carboxymethyl-dextran modified magnetic nanoparticle immobilized TGM. The results of the TGM stability study at 50 C also confirmed higher activity and better stability of CMD-MNPs compared to the activity and stability of CMD-oleic. An activity and stability study of free and immobilized TGM at 70 C (Fig. 9) gave surprisingly different results than those obtained at 50 C. The activity of immobilized TGM decreased after 2 h (the exception is when CMD-oleic is incubated in a non-aqueous medium), but with further prolongation of exposure to 70 C, an
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
9
Table 1 Productivity of non.-immobilized and immobilized TGM after incubation at 50 C. The product was hydroxamate. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.) Sample
Activity [U/mL
free TGM 0h free TGM 6h CMD-MNPs with buffer 0h CMD-MNPs with buffer 6h CMD-MNPs without buffer 0h CMD-MNPs without buffer 6h CMD-oleic with buffer 0h CMD-oleic with buffer 6h CMD-oleic without buffer 0h CMD-oleic without buffer 6h
0.0977 0.0033 0.0316 0.0113 0.0314 0.0201 0.0282 0.0106 0.0446 0.0260
increase in the activity of immobilized enzyme was observed. This increase could be attributed to the decomposition of CMD-MNPs or CMD-oleic and as a result, TGM comes in contact with some cations (Kþ and Co2þ) contained in CMD-MNPs, which, according to the literature (Kieliszek and Misiewicz, 2014), increase the activity of TGM. These results confirm better stability of CMD-MNPs compared to CMD-oleic. When non-immobilized TGM was incubated at 70 C, the productivity, when hydroxamate was a product, decreased even more than when incubated at 50 C, from 120.15 mg/(min mgenz) to 0.21 mg/(min mgenz) after 6 h of incubation, a 99% reduction. As expected, the productivity of the immobilized TGM was much higher. Here again, we see that in a non-aqueous medium, the immobilized TGM was more stable than in an aqueous medium (Table 2). But still, when CMD-oleic was incubated for 6 h at 70 C in an aqueous medium, its productivity dropped from initial 149.01 mg/(min mgenz) to 112.88 mg/(min mgenz), or 25%, which is much less than the productivity decrease of the non-immobilized enzyme. In the other three cases, a productivity increase was observed after 6 h of incubation at 70 C. 3.1.8. Reusability of immobilized TGM For practical processes, it is important that immobilized TGM preserve high catalytic activity and a maximum number of reaction cycles and at the same time ease recovery for reuse (Tesfaw and Assefa, 2014). Stability of immobilized enzymes can increase or decrease depending on the type of matrix, as well as on the interaction between the enzyme and the matrix (Dwevedi, 2016). Therefore, operational stability of immobilized TGM was evaluated through its repeated use. The experiments were performed using carriers for TGM immobilization: CMD-MNPs and CMD-oleic, prepared under optimal conditions. From Fig. 10, it can be observed that by increasing the number of cycles, where TGM was reused, activity of immobilized TGM decreased, as was expected. However, CMD-MNPs and CMD-oleic retained 100% activity after three
enzyme]
Productivity [mg/(min mg
enzyme)]
111.88 3.78 152.25 54.44 151.93 97.25 65.02 24.44 130.20 75.90
reaction cycles. A slight decrease in immobilized TGM activity was observed after the fourth reaction cycle, but the residual activity was still higher than 95% for both carriers. Immobilized TGM retained 68% of its initial activity after six reaction cycles when it was immobilized onto CMD-MNPs and 63% when it was immobilized onto the CMD-oleic. The decrease in activity over the cycles can be caused by the gradual inactivation of the immobilized TGM. Desorption of the enzyme throughout the cycles was not expected, since the enzyme is covalently attached to the support (Fortes et al., 2017). This was also confirmed with the Bradford test determining the protein concentration. Enzymes that are attached by various covalent bonds either directly or by activated matrix are found to be very stable with high reusability (Dwevedi, 2016). Compared to the case when TGM was immobilized onto a polyethersulfone membrane surface and the enzymatic membrane activity was sharply decreased after three regeneration cycles (Fortes et al., 2017), the stability of TGM via immobilization on CMD-MNPs and CMD-oleic was significantly improved. Again, CMD-MNPs gave higher productivity than CMD-oleic after 6 reaction cycles. The results also show that the immobilized enzyme is quite stable on the surface of the nanocomposite, since the half-life of the immobilized TGM was not achieved even after the sixth reaction cycle. Consequently, by using an immobilized TGM, a significant reduction in operating costs could be achieved. 4. Conclusion The TGM enzyme was successfully immobilized onto CMDMNPs and CMD-oleic prepared by co-precipitation of iron salts in the presence of a base. This kind of immobilized TGM allows easy recovery and reuse by a simple magnetic separation. Under optimal conditions, the enzyme was hyperactivated and showed 110% residual activity for CMD-MNPs and slightly lower (103%) for CMDoleic. The immobilized enzyme presented much higher residual
Table 2 Productivity of non.-immobilized and immobilized TGM after incubation at 70 C. The product was hydroxamate. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.) Sample
Activity [U/mL
free TGM 0h free TGM 6h CMD-MNPs with buffer 0h CMD-MNPs with buffer 6h CMD-MNPs without buffer 0h CMD-MNPs without buffer 6h CMD-oleic with buffer 0h CMD-oleic with buffer 6h CMD-oleic without buffer 0h CMD-oleic without buffer 6h
0.1122 0.0002 0.0268 0.0457 0.0229 0.0270 0.0165 0.0125 0.0144 0.0236
enzyme]
Productivity [mg/(min mg 120.15 0.21 177.71 303.03 151.93 323.09 149.01 112.88 130.20 257.68
enzyme)]
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
10
Fig. 10. Residual activity of immobilized TGM as a function of repetitive use. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
thermal stability compared to the free enzyme, and even after the sixth catalysis cycle, the half-life of the immobilized TGM was not achieved. Taking into consideration all results, CMD-MNPs or CMDoleic proved to be a suitable and efficient support for immobilization of TGM and has the potential to be used for biosensors and for cleaner production in different real life industrial applications, such as in leather, textile and wool industry. The lifetime of the reaction system could be increased with the high binding strength associated with covalent attachment of TGM onto CMD supports, as could the longevity of immobilized TGM. Conflicts of interest The authors declare no conflict of interest. Author agreement/declaration We certify that all authors have seen and approved the final version of the manuscript being submitted. The article is the authors' original work, has not received prior publication and is not under consideration for publication elsewhere. Acknowledgements The authors acknowledge financial support from the Slovenian Research Agency (research core funding No. P2-0046 - “Separation Processes and Product Design”). References Ahmad, R., Sardar, M., 2015. Enzyme immobilization: an overview on nanoparticles as immobilization matrix. Biochem. Anal. Biochem. 0, 1e8. https://doi.org/10. 4172/2161-1009.1000178. Ali, A., Zafar, H., Zia, M., Ul Haq, I., Phull, A.R., Ali, J.S., Hussain, A., 2016. Synthesis, characterization, applications, and challenges of iron oxide nanoparticles. Nanotechnol. Sci. Appl. 9, 49e67. https://doi.org/10.2147/NSA.S99986. Bardini, G., Boukid, F., Carini, E., Curti, E., Pizzigalli, E., Vittadini, E., 2018. Enhancing
dough-making rheological performance of wheat flour by transglutaminase and vital gluten supplementation. LWT 91, 467e476. https://doi.org/10.1016/j. lwt.2018.01.077. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. https://doi.org/10.1016/0003-2697(76)90527-3. Chaudhary, R., Pati, A., 2016. Purification of protein hydrolyzate recovered from chrome tanned leather shaving waste. J. Am. Leather Chem. Assoc. 2016 10e16. Request PDF. Cortez, J., Bonner, P.L.R., Griffin, M., 2005. Transglutaminase treatment of wool fabrics leads to resistance to detergent damage. J. Biotechnol. 116, 379e386. https://doi.org/10.1016/j.jbiotec.2004.12.007. Dwevedi, A., 2016. Enzyme Immobilization: Advances in Industry, Agriculture, Medicine, and the Environment. Springer International Publishing. Eldin, M.M., EL-Aassar, M.R., EL-Zatahry, A.A., EL-Sabbah, M.M.B., 2015. b-Galactosidase immobilization onto poly (acrylonitrile-Co-methyl methacrylate) nanoparticles. Trends Biomater. Artif. Organs 29, 193e200. Ferk, G., Ban, I., Stergar, J., Makovec, D., Hamler, A., Jagli ci c, Z., Drofenik, M., 2012. A facile route to the synthesis of coated maghemite nanocomposites for hyperthermia applications. Acta Chim. Slov. 59, 366e374. Fortes, C.C.S., Daniel-da-Silva, A.L., Xavier, A.M.R.B., Tavares, A.P.M., 2017. Optimization of enzyme immobilization on functionalized magnetic nanoparticles for laccase biocatalytic reactions. Chem. Eng. Process. Process Intensif. 117, 1e8. https://doi.org/10.1016/j.cep.2017.03.009. es-Favoni, S.P., 2015. Action of microbial transglutaminase Gaspar, A.L.C., de Go (MTGase) in the modification of food proteins: a review. Food Chem. 171, 315e322. https://doi.org/10.1016/j.foodchem.2014.09.019. Hong, R.Y., Feng, B., Chen, L.L., Liu, G.H., Li, H.Z., Zheng, Y., Wei, D.G., 2008. Synthesis, characterization and MRI application of dextran-coated Fe3O4 magnetic nanoparticles. Biochem. Eng. J. 42, 290e300. https://doi.org/10.1016/j.bej.2008. 07.009. Ijima, H., Hou, Y.-T., Takei, T., 2010. Development of hepatocyte-embedded hydrogel-filled macroporous scaffold cultures using transglutaminase. Biochem. Eng. J. 52, 276e281. https://doi.org/10.1016/j.bej.2010.09.003. Kieliszek, M., Misiewicz, A., 2014. Microbial transglutaminase and its application in the food industry. A review. Folia Microbiol. (Praha) 59, 241e250. https://doi. org/10.1007/s12223-013-0287-x. Lenting, H.B.M., Schroeder, M., Guebitz, G.M., Cavaco-Paulo, A., Shen, J., 2006. New enzyme-based process direction to prevent wool shrinking without substantial tensile strength loss. Biotechnol. Lett. 28, 711e716. https://doi.org/10.1007/ s10529-006-9048-0. Makovec, D., Gyergyek, S., Primc, D., Plantan, I., 2015. The formation of magnetic carboxymethyl-dextrane-coated iron-oxide nanoparticles using precipitation from an aqueous solution. Mater. Chem. Phys. 153, 376e383. https://doi.org/10. 1016/j.matchemphys.2015.01.028. Mehta, R.V., Upadhyay, R.V., Charles, S.W., Ramchand, C.N., 1997. Direct binding of
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218 protein to magnetic particles. Biotechnol. Tech. 11, 493e496. https://doi.org/10. 1023/A:1018457915380. Moriyama, K., Minamihata, K., Wakabayashi, R., Goto, M., Kamiya, N., 2013. Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol). Biochem. Eng. J. 76, 37e42. https://doi.org/10.1016/ j.bej.2013.04.007. Motoki, M., Seguro, K., 1998. Transglutaminase and its use for food processing. Trends Food Sci. Technol. 9, 204e210. https://doi.org/10.1016/S0924-2244(98) 00038-7. Na, Z.G., Wang, X.F., Ma, Y.Q., Qian, L., Yan, H.Y., 2012. Research of immobilization of transglutaminase on polypropylene microporous membranes. Adv. Mater. Res. 341-342, 338e344 [WWW Document]. https://doi.org/10.4028/www.scientific. net/AMR.341-342.338. Netto, C.G.C.M., Toma, H.E., Andrade, L.H., 2013. Superparamagnetic nanoparticles as versatile carriers and supporting materials for enzymes. J. Mol. Catal. B Enzym. 85e86, 71e92. https://doi.org/10.1016/j.molcatb.2012.08.010. Rachel, N.M., Pelletier, J.N., 2013. Biotechnological applications of transglutaminases. Biomolecules 3, 870e888. https://doi.org/10.3390/biom3040870. Ramamurthy, G., Krishnamoorthy, G., Sastry, T.P., Mandal, A.B., 2014. Rationalized method to enhance the chromium uptake in tanning process: role of Gallic acid. Clean Technol. Environ. Policy 16, 647e654. https://doi.org/10.1007/s10098013-0651-1. Romeih, E., Walker, G., 2017. Recent advances on microbial transglutaminase and dairy application. Trends Food Sci. Technol. 62, 133e140. https://doi.org/10. 1016/j.tifs.2017.02.015. Shi, Y.-G., Qian, L., Zhang, N., Han, C.-R., Liu, Y., Zhang, Y.-F., Ma, Y.-Q., 2011. Changes in morphology and activity of transglutaminase following cross-linking and immobilization on a polypropylene microporous membrane. Mol. Basel Switz. 16, 10046e10058. https://doi.org/10.3390/molecules161210046. Silambarasan, S., Aravindhan, R., Rao, J.R., Thanikaivelan, P., 2015. Waterless tanning: chrome tanning in ethanol and its derivatives. RSC Adv. 5, 66815e66823. https://doi.org/10.1039/C5RA11740B. Taylor, M.M., Marmer, W.N., Brown, E.M., 2007. Evaluation of polymers prepared from gelatin and casein or whey as potential fillers. J. Am. Leather Chem. Assoc. Tesfaw, A., Assefa, F., 2014. Applications of transglutaminase in textile, wool, and
11
leather processing. Int. J. Text. Sci. 3, 64e69. Torchilin, V.P., 1991. Immobilized Enzymes in Medicine. Springer-Verlag Berlin And Heidelberg Gmbh & Company K. Wang, W., Han, X., Yi, H., Zhang, L., 2018. The ultrafiltration efficiency and mechanism of transglutaminase enzymatic membrane reactor (EMR) for protein recovery from cheese whey. Int. Dairy J. 80, 52e61. https://doi.org/10.1016/j. idairyj.2017.12.012. Xu, J., Sun, J., Wang, Y., Sheng, J., Wang, F., Sun, M., 2014. Application of iron magnetic nanoparticles in protein immobilization. Mol. Basel Switz. 19, 11465e11486. https://doi.org/10.3390/molecules190811465. Yang, D., Wang, Xueyan, Shi, J., Wang, Xiaoli, Zhang, S., Han, P., Jiang, Z., 2016. In situ synthesized rGOeFe3O4 nanocomposites as enzyme immobilization support for achieving high activity recovery and easy recycling. Biochem. Eng. J. Part A 273e280. https://doi.org/10.1016/j.bej.2015.10.003. Yang, M.-T., Chang, C.-H., Wang, J.M., Wu, T.K., Wang, Y.-K., Chang, C.-Y., Li, T.T., 2011. Crystal structure and inhibition studies of transglutaminase from streptomyces mobaraense. J. Biol. Chem. 286, 7301e7307. https://doi.org/10.1074/jbc.M110. 203315. Yu, C.-M., Zhou, H., Zhang, W.-F., Yang, H.-M., Tang, J.-B., 2016. Site-specific, covalent immobilization of BirA by microbial transglutaminase: a reusable biocatalyst for in vitro biotinylation. Anal. Biochem. 511, 10e12. https://doi.org/10.1016/j.ab. 2016.07.026. Zhang, D., Zhu, Y., Chen, J., 2010. Microbial transglutaminase production: understanding the mechanism. Biotechnol. Genet. Eng. Rev. 26, 205e222. Zhou, J.Q., He, T., Wang, J.W., 2016. The microbial transglutaminase immobilization on carboxylated poly(N-isopropylacrylamide) for thermo-responsivity. Enzym. Microb. Technol. 87e88, 44e51. https://doi.org/10.1016/j.enzmictec.2016.02. 012. Zhu, Y., Tramper, J., 2008. Novel applications for microbial transglutaminase beyond food processing. Trends Biotechnol. 26, 559e565. https://doi.org/10.1016/j. tibtech.2008.06.006. Zhu, Z., Liu, L., Gong, D., 2013. Transglutaminase-mediated restoration of historic silk and its ageing resistance. Herit. Sci. 1, 13. https://doi.org/10.1186/20507445-1-13.
Contents lists available at ScienceDirect
Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Enhanced activity of immobilized transglutaminase for cleaner production technologies Marjetka Gajsek a, b, Urska Jan ci c a, Katja Vasi c a, Zeljko Knez a, c, Maja Leitgeb a, c, * a Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova ulica 17, SI2000 Maribor, Slovenia b Kozmetika Afrodita d.o.o., Kidriceva 54, SI-3250, Rogaska Slatina, Slovenia c Faculty of Medicine, University of Maribor, Smetanova 17, SI-2000, Maribor, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 April 2019 Received in revised form 26 August 2019 Accepted 28 August 2019 Available online 3 September 2019
The enzyme transglutaminase (TGM) has a very important role in applications in various industries. Potentially, a substrate for microbial TGM may be any proteinaceous molecule with glutamine and lysine residues, which broadens the importance of microbial TGM for clean industrial applications or for the use of biosensors. Because stabilization and reusability of an enzyme is an economically important goal for enzymatic processes, the purpose of this study was immobilization of TGM onto magnetic nanoparticles (MNPs) synthesized by the co-precipitation of Fe2þ in Fe3þ ions and modified with carboxymethyldextran (CMD). Synthesized nanoparticles were firstly activated with a cross-linking agent (glutaraldehyde (GA) and/or pentaethylenehexamine (PEHA)) and then used for covalent immobilization of the enzyme. Activity of immobilized TGM was determined and compared with activity of the free enzyme. Under optimal immobilization conditions, the enzyme was hyperactivated and showed 99% immobilization efficiency and 110% residual activity. The immobilized TGM showed excellent thermal stability at 50 C and 70 C in comparison with non-immobilized enzyme and very good reusability and can be used for cleaner production in different real life industrial applications, such as in leather, textile and wool industry. © 2019 Elsevier Ltd. All rights reserved.
Handling editor: Prof. Jiri Jaromir Klemes Keywords: Immobilization Magnetic nanoparticles Transglutaminase Enzyme stabilization Cleaner technologies
1. Introduction Transglutaminases (TGM, EC 2.3.2.13) are enzymes that have attracted wide interest from both scientific and applied points of view (Mehta et al., 1997). They belong to the class of transferases and catalyze an acyl transfer reaction between the g-carboxamide groups of glutamine residue (acyl donor) and a variety of primary amines (acyl acceptors), including the ε-amino group of lysine residues (Motoki and Seguro, 1998). This enzyme polymerizes proteins intra- or intermolecularly and forms bonds with high resistance to proteolytic degradation (Yang et al., 2011). It is a promising biocatalytic alternative to classical organic chemistry for amide bond synthesis (Rachel and Pelletier, 2013; Zhang et al., 2010). TGMs are involved in many physiological functions, such as blood coagulation, wound healing, epidermal keratinization, and
* Corresponding author. Faculty of Medicine, University of Maribor, Smetanova 17, SI-2000, Maribor, Slovenia. E-mail address: [email protected] (M. Leitgeb). https://doi.org/10.1016/j.jclepro.2019.118218 0959-6526/© 2019 Elsevier Ltd. All rights reserved.
stiffening of the erythrocyte membrane (Motoki and Seguro, 1998). TGMs also play a role in stabilization of photosynthetic complexes in the chloroplast, modification of cytoskeletal proteins, abiotic and biotic stresses, aging, and programmed cell death in plants. Currently, they are used extensively in the food industry to affect the structure of protein systems and cause changes leading to improved texture, emulsifying properties, thermal stability, gelation, syneresis and increased water-binding capacity (Gaspar and es-Favoni, 2015). In other sectors, the potential applications de Go of TGM include the building of collagen- or gelatin-based scaffolds for the construction of bioartificial organs, site-specific protein conjugation with DNA in biotechnology and materials science, textile and leather processing (Yang et al., 2011). The enzyme is very unstable if it is dissolved in water, however, and its inactivation is influenced by heating, strong acids and bases, and some organic solvents. All these adverse conditions greatly limit the application of the enzyme (Na et al., 2012). Such limitations could be eliminated by using TGM in an immobilized form. The immobilized enzyme could be easily separated from the solution, could function in a much wider pH and temperature range, should have higher
2
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
thermal stability compared to the free enzyme, and it could also be reused. However, immobilization can reduce the catalytic activity of the enzyme, so optimization of the immobilization method is crucial in the preparation of an efficient and economical biocatalyst (Zhou et al., 2016). TGM has been widely used in the textile, wool and leather industries, since it can be produced at an industrial level easily by simple fermentation (Tesfaw and Assefa, 2014). In the wool industry, TGM repairs the damage caused by treatments in which an array of chemicals and enzymes is employed for scouring, carding, gilling, combing, drafting, spinning and twisting, which are processing steps for wearable clothes (Cortez et al., 2005; Lenting et al., 2006). TGM treatment followed by hydrogen peroxide, protease and ultrasonification shows that TGM can improve the crease resistance of silk fabric and enhance tensile breaking strength caused by pretreatments (Zhu et al., 2013). In the leather industry, filling is one of the essential steps in processing. The most commonly used natural products in these steps are glucose, flour and gum; however, gelatin and casein are also used as effective filling materials. Treatment of casein with TGM results in highly polymerized gelatines which are able to fill the leather. Therefore, with TGM treatment, expensive filling treatment of leather materials could be replaced with inexpensive proteinaceous industrial byproducts (Taylor et al., 2007; Tesfaw and Assefa, 2014; Zhu and Tramper, 2008). Since the leather industry produces many pollutants that are toxic (Chaudhary and Pati, 2016; Ramamurthy et al., 2014; Silambarasan et al., 2015) and are discharged directly into the soil, causing soil pollution, they affect human health through water and food. Immobilized TGM treatment provides “green”, but most importantly environmentally friendly approaches to clean leather and textile production. TGM is also widely known to modify food proteins, and it is a promising tool to improve functional properties of protein-based products, such as gelation properties, solubility, emulsifying capacity, foaming, viscosity and elasticity. Possibilities for TGM application can be found in many dairy products such as milk proteins, cheese products, fermented milks, milk powders, and caseinate products (Romeih and Walker, 2017). Functional properties of foods have been improved using TGM in this way. TGM has also been used in the process of enhancing dough-making rheological performance of wheat flour via vital gluten (VG) supplementation. For this purpose, 4 different levels of TGM were used in the experimental design, where TGM and VG changed dough rheological behaviour, which offers opportunities for bread quality improvement (Bardini et al., 2018). Tissue TGM obtained from guinea pig liver was also used to develop a hepatocyte-embedded hydrogel-filled macroporous scaffold culture technology to establish a fundamental technology for liver tissue technology (Ijima et al., 2010). Microbial TGM was used in the process of biotinylation of enhanced green fluorescent protein and bacterial alkaline phosphatase in order to prepare streptavidin-immobilized hydrogel (Moriyama et al., 2013). With the rapid development of nanotechnology, nano-carriers (nanoparticles, nanofibers, nanotubes, nanosheets) are becoming increasingly attractive for immobilization of enzymes. Among them, MNPs have attracted a great deal of attention because of their unique properties (superparamagnetism, high surface area, large surface-to-volume ratio). Magnetic metal nanoparticles are also used to immobilize enzymes because of the low cost of synthesis and negligible toxicity (Xu et al., 2014; Yang et al., 2016). It has also been found that the stability and activity of enzymes increase if they are immobilized on such materials (Ahmad and Sardar, 2015). However, uncoated iron oxide nanoparticles are generally unstable in strong acidic solutions and undergo leaching (Xu et al., 2014). They also tend to agglomerate as a result of strong magnetic attraction among particles, van der Waals forces and high energy
surface. This could be avoided by surface modification of nanoparticles via loading of other target chemicals or biological materials during or after the synthesis process. Various materials have been used as coating materials in aqueous solutions, for example, oleic acid, PEG, PVA, dextran, starch and chitosan (Ali et al., 2016; Hong et al., 2008; Xu et al., 2014). Among them, carboxymethyl dextran (CMD) has outstanding properties - it is biodegradable, biocompatible and bioactive (Ferk et al., 2012). Compared to pure dextran, CMD has additional carboxyl groups which can be used for the covalent bonding of additional molecules (Makovec et al., 2015). Some studies on TGM immobilization already exist. This enzyme was immobilized onto a polyethersulfonate membrane surface, where enzymatic membrane filtration efficiency and a mechanism for catalysis of protein cross-linking and separation from cheese whey were investigated. In this case, TGM was covalently immobilized and a protein recovery rate of 85% in a TGM enzymatic membrane reactor (EMR) was achieved (Wang et al., 2018). Another study describes overcoming mass transfer limitations of immobilized TGM, where TGM was covalently immobilized on a thermo-responsive carboxylated poly(N-isopropylacrylamide) (pNIPAM). After conjugation to pNIPAM, TGM showed a down shifting optimal temperature as well as better thermo-stability. The pNIPAM-TGM conjugate was sensitive to change in temperature with a low critical solution temperature (LCST) at around 39 C, leading to easy separation of the biocatalyst after the catalysis reaction (Zhou et al., 2016). An easy approach for the production of reusable immobilized recombinant Escherichia coli biotin ligase (BirA) onto amine-modified magnetic microspheres (MMS) via covalent cross-linking was proposed using microbial TGM. The site-specifically immobilized BirA exhibited approximately 95% of the enzymatic activity of the free BirA, without a significant loss in intrinsic activity after 10 rounds of recycling (P > 0.05) (Yu et al., 2016). TGM antigen was also immobilized on polyamidoamine (PAMAM) to form a nanoprobe for sensing specific anti-tissue transglutaminase (anti-TGM) antibodies (immunoglobin A isotype) in human serum. The sensor was highly specific to antitissue transglutaminase antibodies and showed negligible response to non-specific serum proteins. Using cross-linkers, such as GA and PEHA for functionalization of MNPs was widely used, but not in a relation to immobilizing TGM. As there are not many studies based on immobilizing TGM onto magnetic supports, the goal of our study was to optimize immobilization of the TGM onto a magnetic nano-carrier, modified with CMD, which has not yet been widely investigated. The covalent attachment of TGM to the support surface via GA and PEHA functionalization was expected to result in a greater stability of the immobilized TGM. Our goal was to prepare an economical biocatalyst that is non-toxic and biodegradable with high activity, improved stability and reusability, which would reduce the cost of enzyme biocatalysis for industrial applications. Such immobilized TGM would have a possibility to be used for cleaner production in different real life industrial applications, such as in leather, textile and wool industry. First, MNPs were prepared, and in their modified and activated form (using GA and PEHA functionalization), they were used for immobilization of TGM (Probind TXo). Immobilization efficiency and residual activity of immobilized TGM was determined, and thermal stability of immobilized and free TGM and reusability of the immobilized TGM were investigated. 2. Materials and methods 2.1. Reagents and materials Transglutaminase, Probind TXo was obtained from BDF Natural Ingredients, with initial activity of 0.07 U/mL and initial protein
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
concentration of 0.46 mg/mL. Carboxymethyl-dextran (CMD) sodium salt, bovine serum albumin (BSA), egg albumin (EA), glutaraldehyde (GA), pentaethylenehexamine (PEHA), ethanol (95%), LGlutamic acid g-monohydroxamate, L-glutathione reduced, hydroxylamine hydrochloride (99%), trichloroacetic acid and Tris buffer (Trizma base) were purchased from Sigma-Aldrich. Z-Glutaminylglycine (Z-Gln-Gly-OH) was purchased from Zedira GmbH, Germany. Iron (II) chloride tetrahydrate, iron (III) chloride hexahydrate, Coomassie brilliant blue G-250, phosphoric acid (85%), hydrochloric acid (37%) and acetic acid (100%) were purchased from Merck. Ammonia was purchased from Chem-Lab and calcium chloride from Kemika. 2.1.1. Preparation of MNPs modified with CMD MNPs were prepared using a co-precipitation method. In the procedure, Fe2þ (0.101 M) and Fe3þ (0.204 M) ions were dispersed in milliQ water under nitrogen flow with vigorous stirring and the temperature was raised to 85 C. Then, CMD-ammonia solution (5 g dextran per 20 mL H2O and 5 mL 25% NH3) was added in several successive small additions over a period of 20 min. The suspension was aged at 85 C for 1 h and the colour of the suspension changed from brown to black. The cooled suspension was then magnetically decanted and washed several times with milliQ water. Then, prepared surface-modified CMD coated nanoparticles (CMD-MNPs) were dried at 50 C and used for immobilization. 2.1.2. Preparation of CMD-MNPs, modified with citric acid Following the co-precipitation method of Fe2þ and Fe3þ ions and the addition of CMD-ammonia solution (5 g dextran per 20 mL H2O and 5 mL 25% NH3), as described in section 2.2, the solution was vigorously stirred at 85 C for 10 min. After 10 min, 2.5 mL of citric acid (0.5 g/mL) was added dropwise and the solution was vigorously stirred for 90 min at 90 C under nitrogen flow. The cooled suspension was then magnetically decanted and washed several times with milliQ water. The prepared MNPs modified with CMD and citric acid (CMD-citric) were dried at 50 C. 2.1.3. Preparation of CMD-MNPs modified with oleic acid Following the co-precipitation method of Fe2þ and Fe3þ ions and addition of CMD-ammonia solution (5 g dextran per 20 mL H2O and 5 mL 25% NH3), as described in section 2.2, the solution was vigorously stirred at 85 C for 10 min. After 10 min, 3 mL of oleic acid was added dropwise and the solution was vigorously stirred for another 90 min at 90 C under nitrogen flow. The cooled suspension was then magnetically decanted and washed several times with milliQ water. The prepared MNPs, modified with CMD and oleic acid (CMD-oleic), were dried at 50 C. 2.1.4. Immobilization of TGM onto nanoparticles The immobilization was carried out using a GA and/or PEHA chemical activation procedure. Synthesized nanoparticles were stirred in a solution of GA and/or PEHA in a Tris buffer with pH 6. The mixture was shaken at 400 rpm for 2 h at room temperature. The activated nanoparticles were then separated from the supernatant in the presence of a magnetic field and further used for immobilization of TGM. TGM was added to activated MNPs, which were previous dispersed in a Tris buffer with pH 6 and the reaction mixture was shaken for 4 h at 10 C. After immobilization of TGM onto CMD nanoparticles, the unbound enzyme was removed by washing with a Tris buffer with pH 6. Supernatants were separated from nanoparticles using a magnet and further used to determine the concentration of the unbound enzyme and thus the immobilization efficiency. Activity of immobilized TGM was also determined.
3
2.1.5. Protein concentration Protein concentration was quantified with Bradford microcrystalline assay (Bradford, 1976), using BSA as calibration standard. 20 mL of enzyme and 1 mL of Bradford reagent were mixed and measured at 595 nm on UV/VIS spectrophotometer after 15 min incubation at room temperature. Control sample contained equivalent volume of Milli-Q water instead of enzyme solution. Immobilization efficiency was calculated by Equation (1):
Immobilization efficiencyð%Þ ¼
ðci cs Þ *100 ci
(1)
where: ci - concentration of TGM. cs - concentration of unbound TGM, collected in supernatant. Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.
2.1.6. Activity assay of TGM TGM activity was determined by a colorimetric hydroxamate procedure (Fig. 1) in which Z-Gln-Gly was used as the amine acceptor substrate and hydroxylamine as amine donor. In the presence of TGM, hydoxylamine was incorporated into Z-Gln-Gly to form Z-glutamyl-hydroxamate-glycine, which developed a coloured complex with iron (III) detectable at 525 nm. One unit of TGM activity was defined as the amount of enzyme that causes the formation of 1.0 mmole of hydroxamate per minute by catalyzing the reaction between Z-Gln-Gly and hydroxylamine at pH 6.0 at 37 C. L-glutamic acid g-monohydroxamate was used as a standard probe. Activity (U/mL) of the enzyme was calculated according to Equation (2):
Activity
U mL
¼
Atest ð525nmÞ df ðEnM1 Þ ð10Þ ð1; 23Þ
(2)
EnM1 ¼ Atest ð525nmÞ 1:1 where: Atest (525 nm) - Absorbance of sample measured at 525 nm. df - dilution factor. 1.1-volume of standard (in mL). 1.23-volume of colour mix (in mL).
Fig. 1. Calorimetric assay for detection of TGM activity (Makovec et al., 2015).
4
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
10 - time of reaction (in min). Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%. 2.1.7. Thermal stability of free and immobilized TGM To study the thermal stability of free and immobilized TGM, it was exposed to two different temperatures for a defined time in 1 M Tris buffer adjusted to pH 6 and also in nonaqueous pure form without buffer. Temperatures chosen for thermal stability study of TGM were 50 C and 70 C, respectively. Incubation time at a certain temperature was 2 h, 4 h and 6 h. After incubation, the samples were brought to room temperature, substrates were added, and the residual activity of the enzyme was determined as described in section 2.4. The residual activity of TGM was calculated according to the enzyme activity before incubation at different temperatures. 2.1.8. Reusability assay The possibility of immobilized TGM reuse was evaluated through 6 consecutive reaction cycles. In the presence of TGM, hydoxylamine was incorporated into Z-Gln-Gly to form Z-glutamylhydroxamate-glycine. Activity of the immobilized TGM was determined after each reaction cycle by activity test for TGM. At the end of each reaction cycle, immobilized TGM was magnetically separated and washed with 1 M Tris buffer with pH 6 before being reused. The activity of the first cycle was defined as 100% activity of the immobilized TGM. Residual activity (%) was defined as the ratio between the enzyme activity at the end of each cycle and the enzyme activity from the first cycle. 3. Results and discussion 3.1. Optimization of TGM immobilization conditions onto MNPs The success of the immobilization procedure depends particularly upon the choice of the enzyme, the support and the immobilization method (Netto et al., 2013). In order to effectively immobilize TGM and improve enzyme stability, activity and reusability, suitable immobilization conditions are critical. Therefore, five factors affecting immobilization of TGM covalently bound onto MNPs were investigated.
Fig. 2. Effect of shaking speed on the immobilization efficiency and residual activity of TGM. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
feeders and the sequence of their addition were investigated in order to further optimize immobilization and maintain the highest residual TGM activity. It is well known that BSA facilitates enzyme immobilization when high concentrations of glutaraldehyde are detrimental to enzyme activity or when the enzyme has a low content of lysine residues. The carrier was activated either with PEHA, GA or combination of both. Albumins were chosen because they have proven to be effective enzyme stabilizers in some cases. They can stabilize the active enzyme structure by forming new bonds (hydrogen, electrostatic or donor-acceptor) between the enzyme and a carrier matric (Torchilin, 1991). BSA or EA with concentration 50 mg/mL was added at the same time as the enzyme (Fig. 3) and also separately so that at first, stabilizing protein was added and later the enzyme solution (Fig. 4). Other parameters were fixed (1 vol % GA or 10 vol % PEHA or combination of 10% PEHA þ0.25% GA, cross-linking time 2 h, immobilization time 4 h at 10 C). As shown in Figs. 3 and 4, the amount of immobilized enzyme significantly increased with the use of stabilizing proteins. When stabilizing proteins BSA or EA were used
3.1.1. Effect of shaking speed To determine the influence of shaking speed on immobilization efficiency and residual activity of TGM, experiments were performed under the following conditions: 2-h activation of the carrier with 10 vol % PEHA, 4-h immobilization of the mixture 100 mg/mL of TGM and 50 mg/mL of BSA at 10 C, and different shaking speeds (300, 400 or 500 rpm). As shown in Fig. 2, shaking speed does not significantly affect the TGM immobilization efficiency, but this cannot be claimed for residual activity of the enzyme, since it was highest (47%) at 400 rpm and lowest (20%) at 500 rpm. The obtained results confirm the importance of this parameter optimization when an immobilization procedure is introduced for a certain enzyme. It is assumed that at a higher shaking speed (500 rpm), TGM inactivation occurs due to strong shear forces. Inactivation is the result of destabilization of electrostatic, hydrophobic and hydrogen bonds of the enzyme, which leads to irreversible enzymatic denaturation and, consequently, lower residual TGM activity. Therefore, a shaking speed of 400 rpm was selected as optimal and all further experiments were done at this shaking speed. 3.1.2. Effect of stabilizing proteins addition The effect of different albumin proteins (BSA or EA) as proteic
Fig. 3. Effect of stabilizing proteins (BSA or EA): stabilizing protein was added together with the enzyme. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%).
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
together with TGM, immobilization efficiency reached 92% when BSA was used and 87% when EA was used (Fig. 3). When stabilizing proteins BSA or EA were added before the addition of the enzyme, immobilization efficiency was much higher (Fig. 4), and reached 98% when BSA was used and 99% when EA was used. Without BSA or EA, it was only 60% when nano-carrier was activated with a combination of 10 vol % PEHA and 0.25% GA. Moreover, the sequence of stabilizing protein addition is an important parameter that may influence enzyme activity. Indeed, in all cases the activity of immobilized TGM was higher when stabilizing protein was added before the addition of enzyme. From the results, it is clear that using only BSA as proteic feeder increases the activity of immobilized TGM. BSA, which contains 66 lysines in its sequence, increased the content of lysine residues, and the consequent crosslinking efficiency. By adding stabilizing protein BSA before the addition of the enzyme and using 10 vol % PEHA for activation of CMD-carrier, TGM was hyperactivated with a residual activity of 109%. Consequently, these parameters were selected as optimal and used for further optimization. 3.1.3. Effect of the TGM concentration To determine the effect of TGM concentration on immobilization efficiency and residual activity of the enzyme, TGM solutions with different concentrations (75, 100 and 150 mg/mL) were used. Other parameters were fixed (10% PEHA or combination of 10% PEHA þ 0.25% GA, 50 mg/mL BSA, cross-linking time 2 h, immobilization time 4 h at 10 C). As can be seen from Fig. 5, immobilization efficiency and residual activity of the enzyme strongly depend on the concentration of the enzyme. The amount of immobilized enzyme and residual activity increased with the increase of TGM concentration, but only up to 100 mg/mL of TGM. With further increase of TGM concentration, immobilization efficiency decreased. We assume that the enzyme immobilization had already reached its maximum at 100 mg/mL of TGM and the surface of the nano-carrier was saturated with the enzyme. The decrease in activity at high TGM concentration might also be related to the
5
aggregation of the enzyme, which makes some active sites of the enzyme hidden (Cortez et al., 2005). The same effect was observed by Eldin et al. (2015), where high beta-galactosidase concentration led consequently to increase the density of the immobilized enzyme molecules on the particles’ surface which in turn led to the rise of “protein-protein” interaction and a consequent reduction of catalytic activity. Although the presence of GA increases immobilization efficiency at low TGM concentration (75 mg/mL), it significantly reduces the enzyme activity at high TGM concentration (150 mg/ml). This effect comes even more to the point at optimal TGM concentration (100 mg/mL), where residual TGM activity of 110% in the case of using PEHA decreased to 70% in the case when GA was added in combination with PEHA. 3.1.4. Effect of immobilization temperature Another important factor influencing activity of the enzyme is immobilization temperature. Enzyme was immobilized onto a CMD-carrier at room temperature (without cooling) and at 10 C (with cooling). From Fig. 6, it may be observed that immobilization temperature does not influence immobilization efficiency, which is the same with or without cooling. But residual activity of TGM was remarkably higher when the enzyme was immobilized onto a carrier at 10 C. It was 110% when enzyme was immobilized onto CMD with cooling and carrier was activated with 10 vol % PEHA, and only 51% under the same conditions but for immobilization at room temperature. When EA was used as a stabilizing protein and the nano-carrier was activated with 10 vol % PEHA in combination with 0.25 vol % GA, the differences in residual TGM activity were lower e only 19%, but as in the previous case, maximal residual activity was lower as well (70% and 66%). 3.1.5. Effect of nano-carrier TGM was immobilized onto different nano-carriers, CMD-MNPs, CMD-oleic, CMD-citric and aminosilane MNPs, to study the effect of the cover layer of MNPs on TGM activity after immobilization.
Fig. 4. Effect of stabilizing proteins (BSA or EA): first stabilizing protein was added before the addition of enzyme. (Each experiment was performed triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
6
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
Fig. 5. Effect of TGM concentration on the immobilization efficiency and residual activity of immobilized TGM. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Fig. 6. Immobilization efficiency and residual activity of TGM immobilized onto CMD nano-carrier with or without cooling. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Comparison of the residual TGM activity of such prepared biocomposites is shown in Fig. 7. Immobilization of TGM was carried out for 4 h at 10 C on a shaker at 400 rpm and a concentration of the enzyme 100 mg/mL, concentration of BSA or EA 50 mg/mL and 10 vol % PEHA or a combination of 10 vol % PEHA and 0.25 vol % GA. The nano-carriers were first activated with PEHA or the combination of PEHA and GA, then a stabilizing protein (BSA or EA) was
added, and finally TGM was immobilized. The results showed that with the selected parameters, the highest residual activity of the enzyme was achieved with CMD-MNPs (110%) and activation of the nano-carrier with 10 vol % PEHA. Residual activity of TGM immobilized onto CMD-oleic, activated with a combination of 10 vol % PEHA and 0.25 vol % GA, was 103%. The lowest residual activity (22%) was shown by TGM, immobilized on CMD-citric, activated
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
7
Fig. 7. Residual activity of TGM immobilized onto different nano-carriers: CMD-MNPs, CMD-oleic, CMD-citric and aminosilane MNPs. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
with a combination of 10 vol % PEHA and 0.25 vol % GA. For CMDMNPs, CMD-oleic and CMD-citric, the use of the BSA stabilizing protein was more appropriate than the use of EA. Conversely, for aminosilane MNPs, residual activity was higher when using EA protein. The choice of carrier and immobilization conditions therefore has a significant effect on residual TGM activity. 3.1.6. Stability of immobilized TGM Evolution of any new immobilization protocols focuses primarily on percentage enzyme recovery and operational stability. Therefore, the thermal stability of immobilized and nonimmobilized TGM and reusability of immobilized TGM were also studied. 3.1.7. Thermal stability of immobilized TGM Thermal stability of the enzyme is a very important parameter for its industrial applications, because it determines the limits for use and reuse of the enzyme, and therefore process costs (Shi et al., 2011). There is a shift in the optimum temperature of immobilized enzymes towards higher temperatures. During enzyme immobilization, free movement of enzyme molecules is obstructed, even at higher temperatures. Thus, enzyme denaturation is not observed due to protection of amino acids at the active site as well as on the surface (Dwevedi, 2016). In general, covalently immobilized enzymes are more resistant to heat than those in the soluble form. Therefore, thermal stability of TGM immobilized on two different nano-carriers, CMD-MNPs and CMD-oleic, both prepared under optimal conditions (CMD-MNPs: 10 vol % PEHA, 50 mg/mL BSA, 100 mg/mL TGM, CMD-oleic: 10 vol % PEHA þ0.25 vol % GA, 50 mg/ mL BSA, 100 mg/mL TGM, 4-h immobilization of the enzyme at 10 C) was tested and compared with that of pure, nonimmobilized enzyme. TGM was incubated at 50 C and 70 C in Tris buffer, adjusted to pH 6 and, in the pure form, in a non-aqueous medium. As shown in Fig. 8 and Fig. 9, immobilized TGM was less sensitive to temperature change than the free enzyme, which almost completely denaturated after 2 h of incubation at both
temperatures. This confirms better thermal stability of immobilized TGM compared to the free enzyme. At 50 C (Fig. 8), enzyme activity decreases as the time of incubation increases from 2 to 6 h in all cases. The enzyme, immobilized on both carriers, CMD-MNPs and CMD-oleic, maintained almost 40% of its activity after 6-h incubation in the buffer. Residual activity of the immobilized enzyme was even higher when TGM was incubated in a non-aqueous medium. This was expected, since it is well known that water is needed for enzyme molecular changes to reach inactivation. The TGMs’ residual activity in the non-aqueous medium was approximately 60% for CMD-oleic, and 66% for CMD-MNPs. The nonimmobilized enzyme was almost completely inactive after 2-h incubation at 50 C. The results show that the stability of TGM depends on its shape or state. Immobilization results in strengthening of the enzyme structure and provides a protective effect against heat denaturation (Shi et al., 2011). Consequently, immobilized enzyme could work in tougher environments with minimal activity loss. Better stability of the immobilized TGM can also be associated with a multi-point covalent bond between the TGM and the carrier, which prevents changes in enzyme structure at high temperatures, leading to its deactivation. The obtained results are promising for applications of such immobilized TGMs in industrial use. In other words, after incubation at 50 C, the very high productivity of non-immobilized TGM, which was 111.88 mg/(min mg enz), drastically decreased after 6 h of incubation to 3.78 mg/(min mgenz), which is only 3.4% of the initial value. As can be observed in Table 1, initial productivity for CMD-MNPs and CMD-oleic (incubated at 50 C in a buffer or in a non-aqueous medium) was higher than that of the non-immobilized enzyme. We would expect it to be the other way around as a result of diffusion limitations when using immobilized enzyme. Another advantage is that the productivity decrease after 6 h incubation at 50 C for CMD-MNPs and CMDoleic is, compared to non-immobilized TGM, much lower. The 6 h incubation in a non-aqueous medium resulted in a productivity drop to 97.25 mg/(min mgenz) for CMD-MNPs, which is only 64% of the initial productivity.
8
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
Fig. 8. Thermal stability of free and immobilized TGM at 50 C. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Fig. 9. Thermal stability of free and immobilized TGM at 70 C. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
Since it is well known that structural changes that cause inactivation of the enzyme molecule at high temperatures are more intensive in an aqueous medium, the influence of immobilized TGM incubation at elevated temperature on the productivity of the enzyme was studied. The results show that even in an aqueous medium where a significant decrease in the productivity after 6 h incubation at 50 C is expected, this was 54.44 mg/(min mgenz) for CMD-MNPs, which is much higher that of the non-immobilized enzyme. This confirms excellent stabilization of the
carboxymethyl-dextran modified magnetic nanoparticle immobilized TGM. The results of the TGM stability study at 50 C also confirmed higher activity and better stability of CMD-MNPs compared to the activity and stability of CMD-oleic. An activity and stability study of free and immobilized TGM at 70 C (Fig. 9) gave surprisingly different results than those obtained at 50 C. The activity of immobilized TGM decreased after 2 h (the exception is when CMD-oleic is incubated in a non-aqueous medium), but with further prolongation of exposure to 70 C, an
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
9
Table 1 Productivity of non.-immobilized and immobilized TGM after incubation at 50 C. The product was hydroxamate. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.) Sample
Activity [U/mL
free TGM 0h free TGM 6h CMD-MNPs with buffer 0h CMD-MNPs with buffer 6h CMD-MNPs without buffer 0h CMD-MNPs without buffer 6h CMD-oleic with buffer 0h CMD-oleic with buffer 6h CMD-oleic without buffer 0h CMD-oleic without buffer 6h
0.0977 0.0033 0.0316 0.0113 0.0314 0.0201 0.0282 0.0106 0.0446 0.0260
increase in the activity of immobilized enzyme was observed. This increase could be attributed to the decomposition of CMD-MNPs or CMD-oleic and as a result, TGM comes in contact with some cations (Kþ and Co2þ) contained in CMD-MNPs, which, according to the literature (Kieliszek and Misiewicz, 2014), increase the activity of TGM. These results confirm better stability of CMD-MNPs compared to CMD-oleic. When non-immobilized TGM was incubated at 70 C, the productivity, when hydroxamate was a product, decreased even more than when incubated at 50 C, from 120.15 mg/(min mgenz) to 0.21 mg/(min mgenz) after 6 h of incubation, a 99% reduction. As expected, the productivity of the immobilized TGM was much higher. Here again, we see that in a non-aqueous medium, the immobilized TGM was more stable than in an aqueous medium (Table 2). But still, when CMD-oleic was incubated for 6 h at 70 C in an aqueous medium, its productivity dropped from initial 149.01 mg/(min mgenz) to 112.88 mg/(min mgenz), or 25%, which is much less than the productivity decrease of the non-immobilized enzyme. In the other three cases, a productivity increase was observed after 6 h of incubation at 70 C. 3.1.8. Reusability of immobilized TGM For practical processes, it is important that immobilized TGM preserve high catalytic activity and a maximum number of reaction cycles and at the same time ease recovery for reuse (Tesfaw and Assefa, 2014). Stability of immobilized enzymes can increase or decrease depending on the type of matrix, as well as on the interaction between the enzyme and the matrix (Dwevedi, 2016). Therefore, operational stability of immobilized TGM was evaluated through its repeated use. The experiments were performed using carriers for TGM immobilization: CMD-MNPs and CMD-oleic, prepared under optimal conditions. From Fig. 10, it can be observed that by increasing the number of cycles, where TGM was reused, activity of immobilized TGM decreased, as was expected. However, CMD-MNPs and CMD-oleic retained 100% activity after three
enzyme]
Productivity [mg/(min mg
enzyme)]
111.88 3.78 152.25 54.44 151.93 97.25 65.02 24.44 130.20 75.90
reaction cycles. A slight decrease in immobilized TGM activity was observed after the fourth reaction cycle, but the residual activity was still higher than 95% for both carriers. Immobilized TGM retained 68% of its initial activity after six reaction cycles when it was immobilized onto CMD-MNPs and 63% when it was immobilized onto the CMD-oleic. The decrease in activity over the cycles can be caused by the gradual inactivation of the immobilized TGM. Desorption of the enzyme throughout the cycles was not expected, since the enzyme is covalently attached to the support (Fortes et al., 2017). This was also confirmed with the Bradford test determining the protein concentration. Enzymes that are attached by various covalent bonds either directly or by activated matrix are found to be very stable with high reusability (Dwevedi, 2016). Compared to the case when TGM was immobilized onto a polyethersulfone membrane surface and the enzymatic membrane activity was sharply decreased after three regeneration cycles (Fortes et al., 2017), the stability of TGM via immobilization on CMD-MNPs and CMD-oleic was significantly improved. Again, CMD-MNPs gave higher productivity than CMD-oleic after 6 reaction cycles. The results also show that the immobilized enzyme is quite stable on the surface of the nanocomposite, since the half-life of the immobilized TGM was not achieved even after the sixth reaction cycle. Consequently, by using an immobilized TGM, a significant reduction in operating costs could be achieved. 4. Conclusion The TGM enzyme was successfully immobilized onto CMDMNPs and CMD-oleic prepared by co-precipitation of iron salts in the presence of a base. This kind of immobilized TGM allows easy recovery and reuse by a simple magnetic separation. Under optimal conditions, the enzyme was hyperactivated and showed 110% residual activity for CMD-MNPs and slightly lower (103%) for CMDoleic. The immobilized enzyme presented much higher residual
Table 2 Productivity of non.-immobilized and immobilized TGM after incubation at 70 C. The product was hydroxamate. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.) Sample
Activity [U/mL
free TGM 0h free TGM 6h CMD-MNPs with buffer 0h CMD-MNPs with buffer 6h CMD-MNPs without buffer 0h CMD-MNPs without buffer 6h CMD-oleic with buffer 0h CMD-oleic with buffer 6h CMD-oleic without buffer 0h CMD-oleic without buffer 6h
0.1122 0.0002 0.0268 0.0457 0.0229 0.0270 0.0165 0.0125 0.0144 0.0236
enzyme]
Productivity [mg/(min mg 120.15 0.21 177.71 303.03 151.93 323.09 149.01 112.88 130.20 257.68
enzyme)]
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218
10
Fig. 10. Residual activity of immobilized TGM as a function of repetitive use. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ±1%.)
thermal stability compared to the free enzyme, and even after the sixth catalysis cycle, the half-life of the immobilized TGM was not achieved. Taking into consideration all results, CMD-MNPs or CMDoleic proved to be a suitable and efficient support for immobilization of TGM and has the potential to be used for biosensors and for cleaner production in different real life industrial applications, such as in leather, textile and wool industry. The lifetime of the reaction system could be increased with the high binding strength associated with covalent attachment of TGM onto CMD supports, as could the longevity of immobilized TGM. Conflicts of interest The authors declare no conflict of interest. Author agreement/declaration We certify that all authors have seen and approved the final version of the manuscript being submitted. The article is the authors' original work, has not received prior publication and is not under consideration for publication elsewhere. Acknowledgements The authors acknowledge financial support from the Slovenian Research Agency (research core funding No. P2-0046 - “Separation Processes and Product Design”). References Ahmad, R., Sardar, M., 2015. Enzyme immobilization: an overview on nanoparticles as immobilization matrix. Biochem. Anal. Biochem. 0, 1e8. https://doi.org/10. 4172/2161-1009.1000178. Ali, A., Zafar, H., Zia, M., Ul Haq, I., Phull, A.R., Ali, J.S., Hussain, A., 2016. Synthesis, characterization, applications, and challenges of iron oxide nanoparticles. Nanotechnol. Sci. Appl. 9, 49e67. https://doi.org/10.2147/NSA.S99986. Bardini, G., Boukid, F., Carini, E., Curti, E., Pizzigalli, E., Vittadini, E., 2018. Enhancing
dough-making rheological performance of wheat flour by transglutaminase and vital gluten supplementation. LWT 91, 467e476. https://doi.org/10.1016/j. lwt.2018.01.077. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. https://doi.org/10.1016/0003-2697(76)90527-3. Chaudhary, R., Pati, A., 2016. Purification of protein hydrolyzate recovered from chrome tanned leather shaving waste. J. Am. Leather Chem. Assoc. 2016 10e16. Request PDF. Cortez, J., Bonner, P.L.R., Griffin, M., 2005. Transglutaminase treatment of wool fabrics leads to resistance to detergent damage. J. Biotechnol. 116, 379e386. https://doi.org/10.1016/j.jbiotec.2004.12.007. Dwevedi, A., 2016. Enzyme Immobilization: Advances in Industry, Agriculture, Medicine, and the Environment. Springer International Publishing. Eldin, M.M., EL-Aassar, M.R., EL-Zatahry, A.A., EL-Sabbah, M.M.B., 2015. b-Galactosidase immobilization onto poly (acrylonitrile-Co-methyl methacrylate) nanoparticles. Trends Biomater. Artif. Organs 29, 193e200. Ferk, G., Ban, I., Stergar, J., Makovec, D., Hamler, A., Jagli ci c, Z., Drofenik, M., 2012. A facile route to the synthesis of coated maghemite nanocomposites for hyperthermia applications. Acta Chim. Slov. 59, 366e374. Fortes, C.C.S., Daniel-da-Silva, A.L., Xavier, A.M.R.B., Tavares, A.P.M., 2017. Optimization of enzyme immobilization on functionalized magnetic nanoparticles for laccase biocatalytic reactions. Chem. Eng. Process. Process Intensif. 117, 1e8. https://doi.org/10.1016/j.cep.2017.03.009. es-Favoni, S.P., 2015. Action of microbial transglutaminase Gaspar, A.L.C., de Go (MTGase) in the modification of food proteins: a review. Food Chem. 171, 315e322. https://doi.org/10.1016/j.foodchem.2014.09.019. Hong, R.Y., Feng, B., Chen, L.L., Liu, G.H., Li, H.Z., Zheng, Y., Wei, D.G., 2008. Synthesis, characterization and MRI application of dextran-coated Fe3O4 magnetic nanoparticles. Biochem. Eng. J. 42, 290e300. https://doi.org/10.1016/j.bej.2008. 07.009. Ijima, H., Hou, Y.-T., Takei, T., 2010. Development of hepatocyte-embedded hydrogel-filled macroporous scaffold cultures using transglutaminase. Biochem. Eng. J. 52, 276e281. https://doi.org/10.1016/j.bej.2010.09.003. Kieliszek, M., Misiewicz, A., 2014. Microbial transglutaminase and its application in the food industry. A review. Folia Microbiol. (Praha) 59, 241e250. https://doi. org/10.1007/s12223-013-0287-x. Lenting, H.B.M., Schroeder, M., Guebitz, G.M., Cavaco-Paulo, A., Shen, J., 2006. New enzyme-based process direction to prevent wool shrinking without substantial tensile strength loss. Biotechnol. Lett. 28, 711e716. https://doi.org/10.1007/ s10529-006-9048-0. Makovec, D., Gyergyek, S., Primc, D., Plantan, I., 2015. The formation of magnetic carboxymethyl-dextrane-coated iron-oxide nanoparticles using precipitation from an aqueous solution. Mater. Chem. Phys. 153, 376e383. https://doi.org/10. 1016/j.matchemphys.2015.01.028. Mehta, R.V., Upadhyay, R.V., Charles, S.W., Ramchand, C.N., 1997. Direct binding of
M. Gajsek et al. / Journal of Cleaner Production 240 (2019) 118218 protein to magnetic particles. Biotechnol. Tech. 11, 493e496. https://doi.org/10. 1023/A:1018457915380. Moriyama, K., Minamihata, K., Wakabayashi, R., Goto, M., Kamiya, N., 2013. Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol). Biochem. Eng. J. 76, 37e42. https://doi.org/10.1016/ j.bej.2013.04.007. Motoki, M., Seguro, K., 1998. Transglutaminase and its use for food processing. Trends Food Sci. Technol. 9, 204e210. https://doi.org/10.1016/S0924-2244(98) 00038-7. Na, Z.G., Wang, X.F., Ma, Y.Q., Qian, L., Yan, H.Y., 2012. Research of immobilization of transglutaminase on polypropylene microporous membranes. Adv. Mater. Res. 341-342, 338e344 [WWW Document]. https://doi.org/10.4028/www.scientific. net/AMR.341-342.338. Netto, C.G.C.M., Toma, H.E., Andrade, L.H., 2013. Superparamagnetic nanoparticles as versatile carriers and supporting materials for enzymes. J. Mol. Catal. B Enzym. 85e86, 71e92. https://doi.org/10.1016/j.molcatb.2012.08.010. Rachel, N.M., Pelletier, J.N., 2013. Biotechnological applications of transglutaminases. Biomolecules 3, 870e888. https://doi.org/10.3390/biom3040870. Ramamurthy, G., Krishnamoorthy, G., Sastry, T.P., Mandal, A.B., 2014. Rationalized method to enhance the chromium uptake in tanning process: role of Gallic acid. Clean Technol. Environ. Policy 16, 647e654. https://doi.org/10.1007/s10098013-0651-1. Romeih, E., Walker, G., 2017. Recent advances on microbial transglutaminase and dairy application. Trends Food Sci. Technol. 62, 133e140. https://doi.org/10. 1016/j.tifs.2017.02.015. Shi, Y.-G., Qian, L., Zhang, N., Han, C.-R., Liu, Y., Zhang, Y.-F., Ma, Y.-Q., 2011. Changes in morphology and activity of transglutaminase following cross-linking and immobilization on a polypropylene microporous membrane. Mol. Basel Switz. 16, 10046e10058. https://doi.org/10.3390/molecules161210046. Silambarasan, S., Aravindhan, R., Rao, J.R., Thanikaivelan, P., 2015. Waterless tanning: chrome tanning in ethanol and its derivatives. RSC Adv. 5, 66815e66823. https://doi.org/10.1039/C5RA11740B. Taylor, M.M., Marmer, W.N., Brown, E.M., 2007. Evaluation of polymers prepared from gelatin and casein or whey as potential fillers. J. Am. Leather Chem. Assoc. Tesfaw, A., Assefa, F., 2014. Applications of transglutaminase in textile, wool, and
11
leather processing. Int. J. Text. Sci. 3, 64e69. Torchilin, V.P., 1991. Immobilized Enzymes in Medicine. Springer-Verlag Berlin And Heidelberg Gmbh & Company K. Wang, W., Han, X., Yi, H., Zhang, L., 2018. The ultrafiltration efficiency and mechanism of transglutaminase enzymatic membrane reactor (EMR) for protein recovery from cheese whey. Int. Dairy J. 80, 52e61. https://doi.org/10.1016/j. idairyj.2017.12.012. Xu, J., Sun, J., Wang, Y., Sheng, J., Wang, F., Sun, M., 2014. Application of iron magnetic nanoparticles in protein immobilization. Mol. Basel Switz. 19, 11465e11486. https://doi.org/10.3390/molecules190811465. Yang, D., Wang, Xueyan, Shi, J., Wang, Xiaoli, Zhang, S., Han, P., Jiang, Z., 2016. In situ synthesized rGOeFe3O4 nanocomposites as enzyme immobilization support for achieving high activity recovery and easy recycling. Biochem. Eng. J. Part A 273e280. https://doi.org/10.1016/j.bej.2015.10.003. Yang, M.-T., Chang, C.-H., Wang, J.M., Wu, T.K., Wang, Y.-K., Chang, C.-Y., Li, T.T., 2011. Crystal structure and inhibition studies of transglutaminase from streptomyces mobaraense. J. Biol. Chem. 286, 7301e7307. https://doi.org/10.1074/jbc.M110. 203315. Yu, C.-M., Zhou, H., Zhang, W.-F., Yang, H.-M., Tang, J.-B., 2016. Site-specific, covalent immobilization of BirA by microbial transglutaminase: a reusable biocatalyst for in vitro biotinylation. Anal. Biochem. 511, 10e12. https://doi.org/10.1016/j.ab. 2016.07.026. Zhang, D., Zhu, Y., Chen, J., 2010. Microbial transglutaminase production: understanding the mechanism. Biotechnol. Genet. Eng. Rev. 26, 205e222. Zhou, J.Q., He, T., Wang, J.W., 2016. The microbial transglutaminase immobilization on carboxylated poly(N-isopropylacrylamide) for thermo-responsivity. Enzym. Microb. Technol. 87e88, 44e51. https://doi.org/10.1016/j.enzmictec.2016.02. 012. Zhu, Y., Tramper, J., 2008. Novel applications for microbial transglutaminase beyond food processing. Trends Biotechnol. 26, 559e565. https://doi.org/10.1016/j. tibtech.2008.06.006. Zhu, Z., Liu, L., Gong, D., 2013. Transglutaminase-mediated restoration of historic silk and its ageing resistance. Herit. Sci. 1, 13. https://doi.org/10.1186/20507445-1-13.