J. Microbiol. Biotechnol. (2010), 20(3), 513–517 doi: 10.4014/jmb.0906.06048 First published online 5 December 2009 Immobilization on Chitosan of a Thermophilic Trehalose Synthase from Thermus thermophilus HJ6 Kim, Hyun-Jung1, Ae-Ran Kim2, and Sung-Jong Jeon1,2,3* 1Department of Biomaterial Control, 2Department of Biotechnology and Bioengineering, and 3Blue-Bio Industry RIC, Dong-Eui University, Busan 614-714, Korea Received: June 24, 2009 / Accepted: September 14, 2009 A thermostable trehalose synthase (TtTSase) from Thermus of the α-1,4-linkage of maltose to the α,α,-1,1-linkage of thermophilus HJ6 was immobilized on chitosan activated trehalose in the absence of coenzyme [23]. This enzymatic with glutaraldehyde. The yield of immobilization was process has the advantages of simple reaction, high substrate evaluated as 39.68%. The optimum pH of the immobilized specificity, high conversion yield, and low cost. Therefore, enzyme was similar to that of the free enzyme. However, it is one of the most efficient processes for the production the optimal temperature ranges were shifted by about 4oC of trehalose in the point of industrial application. owing to better thermal stability after immobilization. The immobilization of enzyme has been often regarded The half-life of heat inactivation for free and immobilized preparative for commercial applications owing to low stability enzymes was 5.7 and 6.3 days at 70oC, respectively, thus [6]. Advantages of immobilized enzymes are that the showing a lager thermostability of the immobilized enzyme. generally expensive biocatalysts can be used repeatedly in When tested in batch reaction, the immobilized enzyme batch or continuous reactors. Enzymes can be immobilized retained its relative activity of 53% after 30 reuses of on various supports and by different methods. Recently, reaction within 12 days, and still retained 82% of its initial immobilization of recombinant TSase for trehalose production activity even after 150 days at 4oC. A packed-bed bioreactor has been reported [4]. The recombinant TSase was with immobilized enzyme showed a maximum yield of 56% immobilized with a high yield onto commercially available trehalose from 100 mM maltose in a continuous recycling Eupergit C250L, an epoxy-activated acrylic bead. In our system (bed volume: 10 ml) under conditions of pH 7.0 studies, chitosan was selected as the support for enzyme and 70oC. immobilization. Chitosan is a natural polyaminosaccharide N Keywords: Chitosan, immobilization, trehalose synthase, obtained by -deacetylation of chitin, and is non-toxic, Thermus thermophilus, thermostability, bioreactor biocompatible, and biodegradable [2, 19]. Its reactive amino and hydroxyl groups after chemical modifications make possible the coupling of enzymes [10]. Therefore, chitosan has been used as a support material for immobilization of Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is enzymes [3, 22]. a nonreducing disaccharide that is widely present in In a previous study, we have cloned and analyzed the various organisms, including bacteria, yeast, fungi, insects, expression of the gene encoding a hyperthermostable invertebrates, and plants [7, 21]. The nonreducing disaccharide trehalose synthase from Thermus thermophilus HJ6 and serves as a carbohydrate reserve and a protecting agent studied its molecular and enzymatic properties [9]. In this against a variety of environmental stresses such as heat, paper, we report the optimization of TSases immobilization cold, desiccation, dehydration, and osmotic pressure [1]. on porous chitosans, the properties of the immobilized These functions have been applied to the development of preparation, and the performance of a bioreactor for trehalose additives, stabilizers, and sweeteners, which are useful in production at high temperature. the food, cosmetic, and pharmaceutical industries [15, 18, 20]. Trehalose synthase (TSase; E.C. 5.4.99.16) converts maltose into trehalose by an intramolecular rearrangement MATERIALS AND METHODS Chemicals, Strains, and Plasmids *Corresponding author Phone: +82-51-890-2278; Fax: +82-51-890-2632; Chitosan (C3646, from crab shells), Tween 80, sodium tripolyphosphate, E-mail: [email protected] Triton X-100, and glutaraldehyde were obtained from Sigma Co. 514 Kim et al. (St. Louis, MO, U.S.A.). All chemicals were of analytical purity and used as purchased. The column (XK16/20) for the packed bed bioreactor was purchased from GE Healthcare. E. coli DH5α and BL21 (DE3) codon plus (Novagen, Inc., San Diego, CA, U.S.A.) were used as the cloning and expression host cells, respectively. The plasmid pJLA503 was preserved in our laboratory as the expression vector for the TtTS gene. Chitosan Particle Preparation Chitosan particles were prepared by a precipitation method [3]. Chitosan was solubilized in 2% acetic acid to a final concentration of 2% (w/v). The solution was filtered through Whatman GF/A filters and added Fig. 1. Scheme of the packed-bed immobilized enzyme reactor. dropwise through a capillary into a gently stirred coagulation liquid 1, Substrate reservoir; 2, piston pump; 3, thermostat column reactor; 4, [1 N sodium hydroxide and 26% (v/v) ethanol]. The obtained particles water circular; 5, hot waterbath. were centrifuged and washed with distilled water until neutrality. The mean size of the particles was determined from optical micrographs taken with an Eclipse 50i optical microscope (Nikon, Japan). One unit of TtTSase activity was defined as the amount of enzyme that catalyzed the formation of 1 µmole of trehalose per minute Enzyme Immobilization under the reaction conditions. Trehalose synthase (TtTSase) from Thermus thermophilus HJ6 cloned in E. coli was produced as previously described [8, 9]. Immobilization Continuous Conversions in a Packed-Bed Reactor on chitosan particles was carried out adding 1 g (wet weight) particles The chitosan particles with TtTSase bound were packed into a XK to a mixture of 3.5 ml of 0.02 mol/l potassium phosphate buffer 16/20 glass column (16×200 mm) (GE Healthcare, Uppsala, Sweden) (pH 7.0) and 1.5 ml of TtTSase (10 mg/ml). The mixtures were gently equipped with a jacket for constant temperature operation. The o shaken at 100 rpm for 2 h, and after a further 16 h at 25oC in the reaction temperature was maintained at 70 C using a water circular presence of 2.5% (v/v) glutaraldehyde, the mixture was centrifuged at (Jeio Tech CW-05G, Korea). The continuous production was carried 12,000 rpm for 2 min. The immobilized enzymes were washed with out using a packed-bed bioreactor (16×100 mm) containing 8.6 g 0.02 mol/l potassium phosphate buffer (pH 7.0) until protein was no (dry weight) of immobilized enzyme preparation. The reactor was longer detected in the washing solution. Protein concentration was filled with 10 ml of immobilized enzyme resin and the working determined using a Bio-Rad protein assay system (Bio-Rad, Hercules, volume (Vw) was evaluated as 10 ml. The column was continuously CA, U.S.A.) with bovine serum albumin as the standard. The fed from the bottom with 100 mM maltose solution in 50 mM immobilized protein amount was calculated by subtracting the sodium phosphate buffer (pH 7.0) at a flow rate of 0.5 ml/min using protein recovered in the supernatant from the protein subjected to a piston pump (Fig. 1). The samples at the outlet of the reactor were immobilization. The immobilization yield was calculated as the ratio taken and used for further analyses. Two duplicates were made for of immobilized protein to protein subjected to immobilization. each determination. Assay of Enzyme Activity Enzyme activity was assayed by measuring trehalose produced from RESULTS AND DISCUSSION 50 mM maltose solution. Maltose solution in 50 mM potassium phosphate buffer (pH 7.0) was incubated with free or immobilized Immobilization of TtTSase o enzyme at 70 C for 30 min in a water bath. The reaction mixture Chitosan is a suitable biocompatible and inexpensive matrix was boiled for 10 min to stop the reaction. The trehalose produced for a bioreactor in which the main features of the free was analyzed by HPLC (Waters Alliance 2690). Chromatography enzyme are preserved. Furthermore, it has been used as a was performed using a carbohydrate analysis column (Waters, support material for immobilization of thermostable enzymes 300 mm×3.9 mm) on a Waters Alliance 2690 system and a Waters model 410 detector. Separation of the samples was accomplished [5, 16]. In view of the thermostable and practicable industrial with a linear gradient using eluant (acetonitrile:water, 75:25). The applications, the thermostable trehalose synthase (TtTSase) column temperature was set at 25oC. A Pierce standard amino acid was immobilized on chitosan. Chitosan supports were mixture (hydrolysis standard) was used to calibrate the analyses. prepared by a precipitation method, and the mean diameter The authentic glucose, maltose, and trehalose were used as controls. of the prepared particles was 2.0 mm. The amount of Table 1. Immobilization yield of support and specific activity of immobilized enzyme. Support Immobilized protein Immobilization yield Immobilized enzyme Specific activity of (mg/mg support) (%) activitya immobilized enzymeb Chitosan 0.16 39.68 0.072 0.45 aUnit of activity: µmol maltose/min/mg dry support. bUnit of specific activity: µmol maltose/min/mg protein. IMMOBILIZATION OF A THERMOPHILIC TREHALOSE SYNTHASE 515 immobilized protein on chitosan particle, the immobilization yield, the activity of TtTSase related to the weight of dry supports as well as the specific activity of immobilized enzyme are summarized in Table 1. The immobilization yield reached 39.68% when the conditions were as follows: coupling time of 18 h at 25oC in 20 mM potassium phosphate buffer (pH 7.0) and enzyme loading of 0.01 g protein/g- supports. The immobilization capacity of chitosan was much lower than that of Eupergit C250L (92% of immobilization yield) that has been previously reported [4]. The specific activities of the free and the immobilized TtTSase were 0.42 and 0.45 U/mg protein, respectively, when measured at pH 7.0 and 70oC.