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Preparative-Scale Kinetic Resolution of Racemic Styrene Oxide by Immobilized Epoxide Hydrolase
S378
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-I.98] Levan from Bacillus subtilis Natto: Optimization of Productivity Using Factorial Design F.C.B. Cabral de Melo, D. Borsato, J.B. Buzato, M.A.P.C. Celligoi ∗ State University of Londrina, Brazil Keywords: Levan; Bacillus subtilis Natto; sucrose; factorial design
Table 1 First factorial design (33 - Box-Behnken) with 3 central points for investigation of the effect of pH (X1 ), temperature (X2 ) and culture time (X3 ) on Productivity (Y1 ) of levan by Bacillus subtilis Natto. Coded Variables
Original Variable
Response
(◦ C)
Runs
X1
X2
X3
pH
T
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
-1 1 -1 1 -1 1 -1 1 0 0 0 0 0 0 0
-1 -1 1 1 0 0 0 0 -1 1 -1 1 0 0 0
0 0 0 0 -1 -1 1 1 -1 -1 1 1 0 0 0
5 7 5 7 5 7 5 7 6 6 6 6 6 6 6
25 25 37 37 31 31 31 31 25 37 25 37 31 31 31
t (hours)
Y1 (g/L. h-1 )
18 18 18 18 12 12 24 24 12 12 24 24 18 18 18
0,35 0,51 0,48 4,80 0,88 1,19 0,39 4,15 1,02 1,96 0,54 3,29 0,66 0,83 0,86
to be studied, B. subtilis Natto, isolated from the Japanese food natto. This strain has shown a great performance in levan production, surpassing many other producers. Thus, taking into account the applications of levan, its value and the lack of work about the production of levan by B.subtilis Natto, in the present study was report the optimization of the levan productivity by B. subtilis Natto by response surface methodology, in order to produce levan on larger scales for future applications. To perform the experiment, the sucrose concentration was fixed in 300 g/L and applied two factorial designs, each with 3 replicates at the central point: the first were composed by three variables at three levels (33 - Box-Behnken), totaling 15 runs, (Table 1). According the ANOVA (␣ = 5%), the pH, temperature and culture time effects were significant and positive, achieving a productivity of 4.80 g/L.h-1 . Thus, the pH was fixed in 7 and a second design was performed, using the same variables in a higher level. This new design was composed with two variables at two levels (22 ), totaling 7 runs (Table 2). Again, both variables effects were significant, but now negative, achieving an optimization of levan productivity, shown in Figure 1, corresponding to 5.20 g/L.h-1 at 32 ◦ C in 18 hours, the lowest levels of the variables, chosen for levan production on larger scales. doi:10.1016/j.jbiotec.2010.09.464 [P-I.99] Preparative-Scale Kinetic Resolution of Racemic Styrene Oxide by Immobilized Epoxide Hydrolase Deniz Yildirim ∗ , Dilek Alagöz, Özlem Alptekin, S.Seyhan Tükel Cukurova University,Sciences and Letters Faculty,Chemistry Department, Turkey Keywords: Epoxide hydrolase; Immobilization; Asymmetric Hydrolysis; Eupergit
Figure 1. Response surface in A) for the effects of temperature and culture time (33 factorial design) in levan productivity; Response surface in B) for the effects of temperature and culture time (22 factorial design) in levan productivity, showing the optimization of the process on the tested conditions.
The microbial polysaccharides are produced and widely studied because their applications. Among of then, highlighted levan, an exopolysaccharide of fructose formed by the linkage of  (2→6) and ramifications  (2→1), with potential application in the food, pharmaceutical and cosmetic industry, because it’s chemical and physiological properties. Bacillus subtilis is a promising levan producer on an industrial scale by ferment sucrose and produce high amounts of levan. Recently, a new variety of this strain began Table 2 Second factorial design (22 ) with 3 central points and fixed pH value in 7, based on data from the first one, for the effect of temperature (X1 ) and culture time (X2 ) on productivity (Y1 ) of levan by Bacillus subtilis Natto. Coded Variables
Original Variable
Runs
X1
X2
T(◦ C)
Response
t (hours)
Y1 (g/L. h-1 )
1 2 3 4 5 6 7
+1 -1 +1 -1 0 0 0
-1 -1 +1 +1 0 0 0
42 32 42 32 37 37 37
18 18 30 30 24 24 24
4,65 5,20 2,80 3,84 4,74 4,59 4,50
Introduction: Enantiopure epoxides and their vicinal diols are recognized as being versatile intermediates for the synthesis of biologically active compounds [1-2].Epoxide hydrolases-catalyzed (EHs, EC 3.3.2.3) enantioselective hydrolysis of one enantiomer of racemic epoxides yields the remaining epoxide and corresponding vicinal diols in an enantiomerically pure form [3-4]. However, it was reported that preparative-scale (100-300 g/L) kinetic resolutions of racemic epoxides catalyzed by EH were limited by the low long-term operational stability of EH, the high-cost associated with their single use [5].The immobilization of enzyme generally offers several advantages such as improvement of enzyme stability, repeated uses of biocatalysts and easy separation of reaction product from biocatalysts. Method: In this study, EH from Aspergillus niger was covalently immobilized onto modified Eupergit C and used preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide.The immobilization procedure consisted of three main steps:pre-treatment of support with 1,2-diaminoethane, glutaraldehyde activation and enzyme coupling. Styrene oxide (SO) and 1-Phenyl-1,2-ethanediol (PED) were analyzed by using HPLC equipped with a Shodex ORpak CDC chiral column and UV-Vis dedector (250 nm). Result: The highest enantiomeric excess of (S)-SO and (R)-PED were determined as 99 and 97%, respectively after 12 h reaction time and yield was about 50%. Enantiomeric ratio (E) was calculated by using the computer program created by Faber [6] and found as >200.Immobilized EH was protected about 90 and 75% of its initial activity at 40 and 60 ◦ C, respectively upon 24 h preincubation.
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
S379
Conclusion: In conclusion, our results showed that the immobilized EH preparation was robust biocatalyst for the preparativescale production of enantiopure SO and PED.
[P-I.101]
doi:10.1016/j.jbiotec.2010.09.465
Semra KOCABIYIK ∗ , Sema AYGAR, Bilsev KOYUNCU
[P-I.100]
Middle East Technical University, Department of Biological Sciences, Turkey Keywords: protein folding; chaperone; heat shock; stability
Evolution of Chymotrypsin-Like Enzymes for Specific Hydrolytic Bioconversions of Industrial Interest Volonté Federica 1,2,∗ , Tessaro Davide 1,2 , Molla Gianluca 1,2 , D’Arrigo Paola 1,2 , Servi Stefano 1,2 , Pollegioni Loredano 1,2 1
Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano and Università degli Studi dell’Insubria, Varese, Italy 2 Flamma S.p.A, via Bedeschi 22, 24040 Chignolo D’isola (BG), Italy Keywords: Chymotrypsin-like; Bioconversion; Site-saturation mutagenesis; Protein engineering In order to possess an efficient hydrolytic enzyme active on various compounds of pharmaceutical interest and to simultaneously overcome the European limitations related to the use of enzymes from animal sources, we planned the production of evolved chymotrypsin-like activities as recombinant proteins in E. coli. Preliminary investigations showed that microbial proteases were most efficiently expressed in E. coli as compared to mammalian ones. In particular the enzyme named MICRO ctr was successfully expressed as a fully soluble and mature, active enzyme. The recombinant enzyme was purified by a single chromatographic (affinity) step; it is active on classical substrates of chymotrypsin such as caseine, azocaseine and N-succinyl-L-phenylalanine-pnitroanilide (SPNA). Noteworthy, MICRO ctr shows a 15-fold lower Km for SPNA as substrate than bovin chymotrypsin. When the recombinant enzyme is used on racemic mixtures of a chiral ␣-substituted phenylpropionate methyl ester a 3-fold faster conversion is observed, as well as a comparable e.e. of the final S-acid product (≈90%) as compared to the commercial pancreatic chymotrypsin from bovin. The active site residues of MICRO ctr putatively involved in the enantioselectivity towards the S-enantiomer were identified by a docking analysis performed using a model of the 3D structure of the enzyme. The position of the side chains belonging to the catalytic triad resembles that observed in chymotrypsin but pocket S1 for substrate binding seems larger in MICRO ctr. Site-saturation mutagenesis was then performed on seven active site residues of MICRO ctr and the libraries were screened on various methyl/ethyl ester derivatives of industrial interest by employing a new colorimetric assay based on the coupling with an alcohol oxidase active on the alcoholic group released by the serine-protease. In conclusion, MICRO ctr (and its evolved variants) represents a suitable alternative to animal chymotrypsin and thus an innovative biotool for biopharmaceutical processes. Project: Metadistretti Regione Lombardia 2007 “Enzimi per Biocatalisi” doi:10.1016/j.jbiotec.2010.09.466
Improving Protein Stability And Enzyme Recovery Under Stress Conditions By Use Of Recombinant Molecular Chaperones
Molecular chaperones are highly conserved stress proteins that play vital roles in mediating polypeptide folding, signalling, chaperoning and cytoprotection inside cells. The major protein folding chaperones Hsp70 (Dna K) and Hsp60 (GroEL) in eucarya and in eubacteria play a central role in the heat shock response of these cells. In most archaea small heat-shock proteins (sHSP) together with thermosomes (possibly in cooperation with the co-factor prefoldin) are the ubiquitous molecular chaperones. Much of the current work is aimed at delineating molecular and cellular mechanisms by which these protein families function in maintaining homeostasis and modulating stress response. Thus, the increased understanding of the chaperoning system enables us to utilize them for enhanced protein quality or quantity control in biotechnological applications. In this study our purpose is to take the advantage of the recombinant expression of stable archaeal chaperones for potential biotechnological applications (e.g. improving the thermostability, disaggregation and proteolysis inhibitions). To this end, we cloned and recombinantly expressed the thermosome (THS), prefoldin (PF), and a small sHSP protein from thermoacidophilic archaeaon Thermoplasma volcanium(Tpv). We showed that these chaperones are both functionally stable and fully functional under stress conditions. Recombinant Tpv THS together with tpvPF reduced the aggregation of chemically denatured citrate synthase from pig heart and also promoted its renaturation in an ATP dependent fashion. The recovery of the enzyme activity was about 5- fold more as compared to spontaneous renaturation. The recombinant sHSP prevented the citrate synthase from aggregating under heat-shock and chemical denaturation conditions. In addition, viability of E. coli cells over expressing tpv sHSP compared with the control culture was enhanced about 15-fold after 60 min at 52 ◦ C. This result indicates that tpv chaperones can be potentially employed for improving the utility and stability of enzymes in various applications, including biochips, bioseparation or diagnostic immunological assays. doi:10.1016/j.jbiotec.2010.09.467 [P-I.102] Efficient Immobilization of Epoxide Hydrolase onto Florisil for Enantioselective Resolution of Racemic Styrene Oxide Deniz Yildirim ∗ , S.Seyhan Tükel, Dilek Alagöz, Özlem Alptekin Cukurova University,Sciences and Letters Faculty,Chemistry Department, Turkey Keywords: Epoxide hydrolase; Racemic epoxide; Kinetic Resolution; Florisil Introduction: Enantiopure epoxides and their vicinal diols are key intermediates in the preparation of bulk drug substances and fine chemicals because of their versatile reactivity [1]. Asymmetric kinetic resolution of racemic epoxide with epoxide hydrolases (EHs; EC 3.3.2.3) gives remaining epoxide and corresponding vicinal diol in an enantiomerically pure form. Of the methods used,
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-I.98] Levan from Bacillus subtilis Natto: Optimization of Productivity Using Factorial Design F.C.B. Cabral de Melo, D. Borsato, J.B. Buzato, M.A.P.C. Celligoi ∗ State University of Londrina, Brazil Keywords: Levan; Bacillus subtilis Natto; sucrose; factorial design
Table 1 First factorial design (33 - Box-Behnken) with 3 central points for investigation of the effect of pH (X1 ), temperature (X2 ) and culture time (X3 ) on Productivity (Y1 ) of levan by Bacillus subtilis Natto. Coded Variables
Original Variable
Response
(◦ C)
Runs
X1
X2
X3
pH
T
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
-1 1 -1 1 -1 1 -1 1 0 0 0 0 0 0 0
-1 -1 1 1 0 0 0 0 -1 1 -1 1 0 0 0
0 0 0 0 -1 -1 1 1 -1 -1 1 1 0 0 0
5 7 5 7 5 7 5 7 6 6 6 6 6 6 6
25 25 37 37 31 31 31 31 25 37 25 37 31 31 31
t (hours)
Y1 (g/L. h-1 )
18 18 18 18 12 12 24 24 12 12 24 24 18 18 18
0,35 0,51 0,48 4,80 0,88 1,19 0,39 4,15 1,02 1,96 0,54 3,29 0,66 0,83 0,86
to be studied, B. subtilis Natto, isolated from the Japanese food natto. This strain has shown a great performance in levan production, surpassing many other producers. Thus, taking into account the applications of levan, its value and the lack of work about the production of levan by B.subtilis Natto, in the present study was report the optimization of the levan productivity by B. subtilis Natto by response surface methodology, in order to produce levan on larger scales for future applications. To perform the experiment, the sucrose concentration was fixed in 300 g/L and applied two factorial designs, each with 3 replicates at the central point: the first were composed by three variables at three levels (33 - Box-Behnken), totaling 15 runs, (Table 1). According the ANOVA (␣ = 5%), the pH, temperature and culture time effects were significant and positive, achieving a productivity of 4.80 g/L.h-1 . Thus, the pH was fixed in 7 and a second design was performed, using the same variables in a higher level. This new design was composed with two variables at two levels (22 ), totaling 7 runs (Table 2). Again, both variables effects were significant, but now negative, achieving an optimization of levan productivity, shown in Figure 1, corresponding to 5.20 g/L.h-1 at 32 ◦ C in 18 hours, the lowest levels of the variables, chosen for levan production on larger scales. doi:10.1016/j.jbiotec.2010.09.464 [P-I.99] Preparative-Scale Kinetic Resolution of Racemic Styrene Oxide by Immobilized Epoxide Hydrolase Deniz Yildirim ∗ , Dilek Alagöz, Özlem Alptekin, S.Seyhan Tükel Cukurova University,Sciences and Letters Faculty,Chemistry Department, Turkey Keywords: Epoxide hydrolase; Immobilization; Asymmetric Hydrolysis; Eupergit
Figure 1. Response surface in A) for the effects of temperature and culture time (33 factorial design) in levan productivity; Response surface in B) for the effects of temperature and culture time (22 factorial design) in levan productivity, showing the optimization of the process on the tested conditions.
The microbial polysaccharides are produced and widely studied because their applications. Among of then, highlighted levan, an exopolysaccharide of fructose formed by the linkage of  (2→6) and ramifications  (2→1), with potential application in the food, pharmaceutical and cosmetic industry, because it’s chemical and physiological properties. Bacillus subtilis is a promising levan producer on an industrial scale by ferment sucrose and produce high amounts of levan. Recently, a new variety of this strain began Table 2 Second factorial design (22 ) with 3 central points and fixed pH value in 7, based on data from the first one, for the effect of temperature (X1 ) and culture time (X2 ) on productivity (Y1 ) of levan by Bacillus subtilis Natto. Coded Variables
Original Variable
Runs
X1
X2
T(◦ C)
Response
t (hours)
Y1 (g/L. h-1 )
1 2 3 4 5 6 7
+1 -1 +1 -1 0 0 0
-1 -1 +1 +1 0 0 0
42 32 42 32 37 37 37
18 18 30 30 24 24 24
4,65 5,20 2,80 3,84 4,74 4,59 4,50
Introduction: Enantiopure epoxides and their vicinal diols are recognized as being versatile intermediates for the synthesis of biologically active compounds [1-2].Epoxide hydrolases-catalyzed (EHs, EC 3.3.2.3) enantioselective hydrolysis of one enantiomer of racemic epoxides yields the remaining epoxide and corresponding vicinal diols in an enantiomerically pure form [3-4]. However, it was reported that preparative-scale (100-300 g/L) kinetic resolutions of racemic epoxides catalyzed by EH were limited by the low long-term operational stability of EH, the high-cost associated with their single use [5].The immobilization of enzyme generally offers several advantages such as improvement of enzyme stability, repeated uses of biocatalysts and easy separation of reaction product from biocatalysts. Method: In this study, EH from Aspergillus niger was covalently immobilized onto modified Eupergit C and used preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide.The immobilization procedure consisted of three main steps:pre-treatment of support with 1,2-diaminoethane, glutaraldehyde activation and enzyme coupling. Styrene oxide (SO) and 1-Phenyl-1,2-ethanediol (PED) were analyzed by using HPLC equipped with a Shodex ORpak CDC chiral column and UV-Vis dedector (250 nm). Result: The highest enantiomeric excess of (S)-SO and (R)-PED were determined as 99 and 97%, respectively after 12 h reaction time and yield was about 50%. Enantiomeric ratio (E) was calculated by using the computer program created by Faber [6] and found as >200.Immobilized EH was protected about 90 and 75% of its initial activity at 40 and 60 ◦ C, respectively upon 24 h preincubation.
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
S379
Conclusion: In conclusion, our results showed that the immobilized EH preparation was robust biocatalyst for the preparativescale production of enantiopure SO and PED.
[P-I.101]
doi:10.1016/j.jbiotec.2010.09.465
Semra KOCABIYIK ∗ , Sema AYGAR, Bilsev KOYUNCU
[P-I.100]
Middle East Technical University, Department of Biological Sciences, Turkey Keywords: protein folding; chaperone; heat shock; stability
Evolution of Chymotrypsin-Like Enzymes for Specific Hydrolytic Bioconversions of Industrial Interest Volonté Federica 1,2,∗ , Tessaro Davide 1,2 , Molla Gianluca 1,2 , D’Arrigo Paola 1,2 , Servi Stefano 1,2 , Pollegioni Loredano 1,2 1
Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano and Università degli Studi dell’Insubria, Varese, Italy 2 Flamma S.p.A, via Bedeschi 22, 24040 Chignolo D’isola (BG), Italy Keywords: Chymotrypsin-like; Bioconversion; Site-saturation mutagenesis; Protein engineering In order to possess an efficient hydrolytic enzyme active on various compounds of pharmaceutical interest and to simultaneously overcome the European limitations related to the use of enzymes from animal sources, we planned the production of evolved chymotrypsin-like activities as recombinant proteins in E. coli. Preliminary investigations showed that microbial proteases were most efficiently expressed in E. coli as compared to mammalian ones. In particular the enzyme named MICRO ctr was successfully expressed as a fully soluble and mature, active enzyme. The recombinant enzyme was purified by a single chromatographic (affinity) step; it is active on classical substrates of chymotrypsin such as caseine, azocaseine and N-succinyl-L-phenylalanine-pnitroanilide (SPNA). Noteworthy, MICRO ctr shows a 15-fold lower Km for SPNA as substrate than bovin chymotrypsin. When the recombinant enzyme is used on racemic mixtures of a chiral ␣-substituted phenylpropionate methyl ester a 3-fold faster conversion is observed, as well as a comparable e.e. of the final S-acid product (≈90%) as compared to the commercial pancreatic chymotrypsin from bovin. The active site residues of MICRO ctr putatively involved in the enantioselectivity towards the S-enantiomer were identified by a docking analysis performed using a model of the 3D structure of the enzyme. The position of the side chains belonging to the catalytic triad resembles that observed in chymotrypsin but pocket S1 for substrate binding seems larger in MICRO ctr. Site-saturation mutagenesis was then performed on seven active site residues of MICRO ctr and the libraries were screened on various methyl/ethyl ester derivatives of industrial interest by employing a new colorimetric assay based on the coupling with an alcohol oxidase active on the alcoholic group released by the serine-protease. In conclusion, MICRO ctr (and its evolved variants) represents a suitable alternative to animal chymotrypsin and thus an innovative biotool for biopharmaceutical processes. Project: Metadistretti Regione Lombardia 2007 “Enzimi per Biocatalisi” doi:10.1016/j.jbiotec.2010.09.466
Improving Protein Stability And Enzyme Recovery Under Stress Conditions By Use Of Recombinant Molecular Chaperones
Molecular chaperones are highly conserved stress proteins that play vital roles in mediating polypeptide folding, signalling, chaperoning and cytoprotection inside cells. The major protein folding chaperones Hsp70 (Dna K) and Hsp60 (GroEL) in eucarya and in eubacteria play a central role in the heat shock response of these cells. In most archaea small heat-shock proteins (sHSP) together with thermosomes (possibly in cooperation with the co-factor prefoldin) are the ubiquitous molecular chaperones. Much of the current work is aimed at delineating molecular and cellular mechanisms by which these protein families function in maintaining homeostasis and modulating stress response. Thus, the increased understanding of the chaperoning system enables us to utilize them for enhanced protein quality or quantity control in biotechnological applications. In this study our purpose is to take the advantage of the recombinant expression of stable archaeal chaperones for potential biotechnological applications (e.g. improving the thermostability, disaggregation and proteolysis inhibitions). To this end, we cloned and recombinantly expressed the thermosome (THS), prefoldin (PF), and a small sHSP protein from thermoacidophilic archaeaon Thermoplasma volcanium(Tpv). We showed that these chaperones are both functionally stable and fully functional under stress conditions. Recombinant Tpv THS together with tpvPF reduced the aggregation of chemically denatured citrate synthase from pig heart and also promoted its renaturation in an ATP dependent fashion. The recovery of the enzyme activity was about 5- fold more as compared to spontaneous renaturation. The recombinant sHSP prevented the citrate synthase from aggregating under heat-shock and chemical denaturation conditions. In addition, viability of E. coli cells over expressing tpv sHSP compared with the control culture was enhanced about 15-fold after 60 min at 52 ◦ C. This result indicates that tpv chaperones can be potentially employed for improving the utility and stability of enzymes in various applications, including biochips, bioseparation or diagnostic immunological assays. doi:10.1016/j.jbiotec.2010.09.467 [P-I.102] Efficient Immobilization of Epoxide Hydrolase onto Florisil for Enantioselective Resolution of Racemic Styrene Oxide Deniz Yildirim ∗ , S.Seyhan Tükel, Dilek Alagöz, Özlem Alptekin Cukurova University,Sciences and Letters Faculty,Chemistry Department, Turkey Keywords: Epoxide hydrolase; Racemic epoxide; Kinetic Resolution; Florisil Introduction: Enantiopure epoxides and their vicinal diols are key intermediates in the preparation of bulk drug substances and fine chemicals because of their versatile reactivity [1]. Asymmetric kinetic resolution of racemic epoxide with epoxide hydrolases (EHs; EC 3.3.2.3) gives remaining epoxide and corresponding vicinal diol in an enantiomerically pure form. Of the methods used,