Microsoft Word - contents_2.doc HUNGARIAN JOURNAL OF INDUSTRIAL CHEMISTRY VESZPRÉM Vol. 31. pp. 71-75 (2003) SOLID-PHASE TRYPTOPHAN SYNTHASE BIOCATALYSTS AND THEIR APPLICATION IN INTEGRATED BIOCONVERSION SYSTEM CS. SISAK, Á. KOVÁCS1, B. SZAJÁNI2, L. BOROSS and L. OROSZ2 (University of Veszprém, Research Institute of Chemical and Process Engineering, P.O.B. 158, H-8201 Veszprém, HUNGARY; sisak@mukki.richem.hu 1University of Veszprém, Department of Chemical Engineering P.O.B. 158, H-8201 Veszprém, HUNGARY 2Eötvös Loránd University, Department of Genetics P.O.B. 120 H-1518 Budapest, HUNGARY) A trpAB gene cloned E. coli strain of high tryptophan-synthase activity was cultivated then the separated biomass was permeabilized [1]. Agglomeration of the cells, entrapment into Ca-alginate gel and adsorption onto Celite-560 beads were investigated to get solid-phase biocatalysts. Biotransformation experiments for production of tryptophan and its 5-hydroxy derivative via coupling L-serine with indole/5-hydroxy indole were carried out in aqueous /organic system. Productivity has been found to the highest in case of using agglomerated biomass and Celite-560 adsorbed biomass but the reusability of the former catalyst was not acceptable. Setting-up possibilities of an integrated system consisting of the tryptophan biosynthesis step and of a product separation step have been studied. The aim was to increase the productivity of the bioprocess through reducing the effect of product inhibition. Adsorption of the product onto XAD-4 neutral resin has proved to be selective enough [2] for its separation from the substrates. The system with alternate operation of the catalytic bioconversion step and the product adsorption step seems to be applicable for production of tryptophan but enhancement of the working stability needs further investigations. Introduction Solid-phase biocatalysts play an important role in enzyme catalytic production of numerous amino acids and their derivatives. If it follows from the character of the process that non-conventional reaction medium should be chosen, the significance of application of immobilized enzymes increases because they are considerably more stable than native enzymes [3] in such conditions. On the other hand, if the efficiency of bioconversion can be enhanced by simultaneous product removal the reusability of the biocatalyst can be improved by its immobilization [2,4]. According to the literature [2,4,5], and to our previous results [6,7], the use of non-aqueous medium and the application of integrated system both have importance in the biocatalytic synthesis of tryptophan and its derivatives: on one hand, indole substrate can be hardly dissolved in aqueous medium, on the other hand, the in situ removal of the formed amino acid is reasonable to decrease the product inhibition. The aim of this article is to show our results in the matter of the biosynthesis carried out by solid- phase biocatalysts produced in different ways as well as concerning the studies on the integrated system including the biocatalysis step and the product removal step. Experimental part Microorganism: Escherichia coli strain with cloned trpAB gene was constructed at the Agricultural Biotechnology Center, Gödöllő, Hungary. 72 Maintenance: The E. coli culture was maintained on solid substrate containing 1 % tryptone, 1 % NaCl, 5% yeast extract, 2-3 % agar, 20 µg/10 cm3 Ampicillin. Fermentation and harvesting: Tubes were washed into 50 cm3 medium in 300 cm3 E. flask, shaken at 37 oC, 190 rpm for 24 h. (Medium: 0.1 mole phosphate buffer pH=7.8 supplemented with 5 g NaCl, 1 g yeast extract, 10 g tryptone, 5 mg Ampicillin in 1 dm3). Then 5 cm3 broth was transferred to 200 cm3 medium and was shaken again for 24 h. A mechanically stirred fermenter (BR-97/INEL) of 5 dm3 was inoculated with 500 cm3 broth, using the aforesaid medium. Cells were harvested by centrifugation then permeabilized by freezing at -20 oC [1]. Preparation of solid-phase biocatalysts: Biocatalyst A: Agglomeration of the cells: The wet, permeabilized biomass was dried on air [6] at room temperature and was stored in refrigerator at 5 oC. Biocatalyst B: Entrapment into Ca-alginate gel: 0.5 g cell mass was suspended in 5.5 cm3 dist. water and the suspension was mixed with 10 cm3 Na-alginate solution of 4 w/w% then it was dropped into 0.5 M CaCl2. Biocatalyst C and Biocatalyst D: Adsorption onto Celite-560 beads: The autoclaved Celite-560 (Fluka) particles (5 g) were added to 200 cm3 fermentation broth at the 15th (C) and the 48th (D) hour of the fermentation, respectively. The separated particles were dried on air [6] at room temperature and were stored in refrigerator at 5 oC. Efficiency of immobilization was evaluated via productivity measurements. The productivity was given in mg Trp / mg fixed protein /h and mg Trp / mg catalyst /h, respectively. Biotransformation conditions: 10 µmol/dm3 pyridoxal-5-phosphate, 0.38 mol/dm3 L-serine, 5 mg/dm3 Ampicillin were solved in dist. water and mixed with cyclohexane containing 0.25 mol/dm3 indol or with 2-etil-hexanol containing 0.25 mol/dm3 5-hidroxy-indole. The volume ratio of aqueous and organic phases was changed between 1:4 and 1:9. Solid-phase biocatalysts (A),(B),(C) or (D) were added with concentration of 0.15-10 mg biomass / cm3 reaction mixture and were shaken in 50-100 cm3 E. flasks at 37 oC for 72-96 h. Concentration of indole or 5-hidroxy- indole in the aqueous phase was 0.05-0.1 M. Adsorption studies: AMBERLITE XAD-4 neutral resin (Rohm and Haas) [2] was used as adsorbent. L-tryptophan or 5-hidroxy-L-tryptophan were dissolved in 0.1 M phosphate buffer and shaken in flasks with the adsorbent at 37 oC and 175 rpm for 1 hour. Studies on parallel adsorption of the substrates and products were carried out in a mixture consisted of 90 % phosphate buffer and 10 % cyclohexane or 2- etil-hexanol. Analytics: Tryptophan and indole content of samples were analyzed by HPLC (HP-1050) UV detector at 254 nm. (Column HYPERSIL 10 µm (250 x 4 mm), eluent: 20:20:60 mixture of acetonitrile, methanol and phosphate buffer (0.0175 M KH2PO4-H3PO4), eluent flow rate 0.7 cm3/min.) Integrated biotransformation – product adsorption system: The equipment for testing the operation of the integrated system (Fig. 1) consisted of 2 parts applied Unit 1 Unit 2 Figure 1: Equipment for testing the operation of the integrated biotransformation - product adsorption system 73 alternately to perform the catalytic bioconversion step and the product adsorption step. Biotrans- formation was carried out in a 500 cm3 E. flask (Unit 1) with 200 cm3 reaction mixture consisted of 20 % organic phase with 0.1 M indole and 80 % aqueous phase with 0.15-0.3 M L-serine. The mixture containing 10 and 5 g biocatalyst B and D, respectively was shaken at 37 oC and 175 rpm. After achieving a sufficiently high amino acid level the reaction mixture was settled then the aqueous phase was transferred into Unit 2 where the product adsorption was performed. The adsorbent, AMBERLITE XAD-4 neutral resin was mixed with the liquid phase at 37 oC, 175 rpm for 1 hour. Then the cyclic functioning of the system carried on and the two-step process was repeated several times in succession. Results Comparison of different types of immobilized biocatalyst: Table 1: Specific tryptophan production of different solid phase biocatalysts of different type during 72 hours reaction Type of immobilized biocatalyst Tryptophan production in72 hours (mg Ttrp /mg protein) Agglomerated permeabilized biomass (Biocatalyst A) 0.157 Ca-alginate entrapped biomass (Biocatalyst B) 0.004 Adsorbed cells onto Celite-560 beads in the 15th hour of fermentation (Biocatalyst C) 0.223 Adsorbed cells onto Celite-560 beads in the 48th hour of fermentation (Biocatalyst D) 0.417 Among the solid-phase biocatalysts, the agglomerated permeabilized biomass showed the best productivity expressed in mg Trp / mg catalyst /h but its reuse was very difficult because – due to the small particle size – the loss was high. Using Ca- alginate entrapped biocatalyst the productivity was low because of the internal diffusional resistance. Cell adsorption onto Celite-560 beads has resulted in sufficient productivity and recycling properties. On the basis of the above presented results, the third type immobilized catalysts were chosen to examine its stability properties, accordingly cells were adsorbed onto Celite-560 beads by method ‘C’ and ‘D’. 0 1 2 3 4 5 6 7 1 2 3 4 5 6 Number of synthesis cycles P ro du ce d L- tr yp to ph an (g /d m 3 ) immobilization in the 15th hour immobilization in the 48 th hour Figure 2: Catalytic stability of Celite-560 supported E. coli cells during tryptophan production cycles The results of stability studies show that the time of addition of the support particles to the fermentation broth influences the catalytic stability of the immobilized cells. If Celite-560 was added in the 15th hour (Biocatalyst C), the initial activity was significantly lower than in the case of the support addition in the 48th hour (Biocatalyst D). On the other hand, the residual activity of Biocatalyst C, left after the 6th cycle was almost two times higher than that of Biocatalyst D. Comparison of formation of tryptophan and 5- hydroxy-tryptophan: As it can be seen in Fig. 3 the kinetics and the productivity of the amino acid formation catalyzed by biocatalyst A are very similar both in case of production of tryptophan and its 5-hydroxy derivative. 74 0 5 10 15 20 25 0 25 50 75 100 125 Reaction time (h) A m in o ac id p ro du ct iv ity (g /c m 3 / g ka t) trp (g/dm3/gkat) 5-OH-trp (g/dm3/gkat) Figure 3: Formation of tryptophan and 5-hydroxy-tryptophan as functions of reaction time, using agglomerated biomass as catalyst Adsorption experiments To develop the adsorption effectiveness we considered the previous results [2] and our selectivity and capacity measurements [7] in aqueous / organic two-phase system as a model of the reaction mixture, which contains the substrates and the product, too. It was found that the capacity of XAD-4 resin adsorbent was considerably lower for tryptophan in the presence of indole than without it. The capacity reducing effect was more pronounced in case of adsorption of 5-hydroxy derivatives (Fig. 4). The presence of L-serine moderated the indole effect in case of the tryptophan [7]. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 0 1 2 3 4 5 6 Initial amino acid concentration (mol/dm3×102) A ds or be d am in o ac id (m ol /g ×1 04 ) trp trp+ind 5-OH-trp 5-OH-trp+5OH-ind Figure 4: Adsorbed L-tryptophan and 5-hydroxy tryptophan quantities on XAD-4 neutral resin as functions of composition of the aqueous liquid phase It was concluded from the adsorption results that the effective tryptophan separation is not possible from the two-phase reaction mixture system. Therefore - after settling of the phases - adsorption experiments were carried out in the aqueous phase saturated with indole. In point of all the facts, the reduced capacity seemed to be high enough to use the adsorbent for on-line tryptophan removal. L-tryptophan production experiments in integrated system The integrated biotransformation - product adsorption system was carried out with alginate entrapped (Biocatalyst A) and Celite-560 adsorbed (Biocatalyst D) cells. The latter catalyst proved to be more applicable because of its higher productivity and better stability. The productivities of the simple biotransformation system and the integrated biotransformation / product adsorption system were compared. The data are summarized in Table 2. It can be seen that efficiency enhancement of 30 % can be achieved using the cyclic integrated system. Table 2: Comparison of the tryptophan productivity of the simple biotransformation system and the integrated biotransformation - product adsorption system Type of solid phase biocatalyst 72 hours tryptophan production in simple biotransforma tion process (g Ttrp / cm3) 72 hours tryptophan production in integrated biotransforma tion / adsorption process (g Ttrp / cm3) Ratio of efficiencies of simple biotransforma tion and integrated systems Ca-alginate entrapped biomass (Biocatalyst B) 0.89 1.12 1.26 Adsorbed cells onto Celite-560 beads in the 15th hour of fermentation (Biocatalyst B) 7.2 9.5 1.32 Conclusion Comparing different methods to produce solid– phase biocatalysts from genetically manipulated E. coli K-12 cells of tryptophan synthase activity it was found that the catalyst immobilized by adsorption on Celite-560 showed the best catalytic character considering both its productivity and stability. 75 Carrying out the bioconversion of indole or 5- hydroxy indole and serine in aqueous/organic reaction mixture it was stated that the kinetics and productivity were very similar both in case of tryptophan and in case of 5-hydroxy-tryptophan formation. 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