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Production and biochemical characterization of microbial transglutaminase of a newly isolated enzyme from Streptomyces spp.
New Biotechnology · Volume 25S · September 2009
water. It is also possible to incorporate other materials, such as, metal particles in the Ion Jelly during its production. Ion Jelly® materials provide compatible microenvironments for enzymes such as oxi-reductases (e.g. HRP, glucose oxidase, among others) recently used in biosensors which showed excellent activity, and high operational and storage stability compared with other successful immobilization methods for the same group of enzymes, making it possible to develop colorimetric/electrochemical biosensors. The direct electron transfer between the immobilized proteins and enzymes and the modified electrodes were already successfully attained and proved using cyclic voltammetry. Besides the immobilization of large molecules, Ion Jelly® has proved to be able to incorporate smaller molecules and to release it slowly, namely with the application of potential variations, as tested with ferrocyanide. The interesting features of the material, together with the low cost of production and incorporation of possible catalysts by replacing small amount of gelatin, one of the constituents of Ion Jelly materials, make it suitable also in the field of new biomaterials for biosensors, either colorimetric and/or electrochemical detection and biofuel cells. doi:10.1016/j.nbt.2009.06.458
2.3.076 Effect of enzyme inactivation on kinetic parameters of immobilized penicillin G acylase in the hydrolysis of penicillin G J. Holtheuer , O. Romero, P. Valencia, A. Illanes, L. Wilson School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2147, Valparaíso, Chile
Enzyme inactivation is a major concern in biocatalytic reactions conducted under harsh process conditions. Kinetic parameters of enzyme biocatalysts are usually considered constant throughout reactor operation, which may not be the case if significant enzyme inactivation has occurred. This may be the case in the synthesis of -lactam antibiotics with penicillin G acylase (PGA), where the use of organic cosolvents is required to displace the equilibrium of the reaction towards synthesis, if the reaction is carried out under thermodynamic control, or to depress the competing hydrolytic reactions, in the case of kinetic control. The present study refers to the effect of partial inactivation of immobilized PGA in organic cosolvents on the kinetic parameters of the biocatalyst. A commercial preparation of E. coli PGA immobilized in methacrylate polymer (Dalenz—PGA) was a kind gift from Dalas Biotech (India) and used throughout this study. Kinetic parameters were determined from initial rate data of penicillin G hydrolysis at different substrate (penicillin G) and products concentrations (6-aminopenicillanic acid, 6APA, and phenyl acetic acid, PAA), determining the corresponding inhibition mechanisms. Inactivation was done at 50 and 85% (v/v) of dioxane down to a residual activity of 10 ± 2% and the kinetic parameters were determined for such inactivated biocatalysts and compared with those of the fresh enzyme. Michaelis constant (Km ) and inhibition constant by PAA (KPAA ) were reduced to 25% of their initial values after inactivation
ABSTRACTS
in dioxane (no significant differences were observed with respect to the dioxane concentration used), while inhibition constant by 6APA (K6APA ) was reduced to 10. Diffusional restrictions of the biocatalysts at reaction conditions cannot explain the magnitude of the differences observed in kinetic parameters that are to a great extent because of structural modifications of the enzyme during inactivation. Significant variation in kinetic parameters occurred as a consequence of enzyme inactivation that should be taken into account when modeling enzyme reactor performance, an aspect to which not much attention has been paid up to now. doi:10.1016/j.nbt.2009.06.459
2.3.077 Production and biochemical characterization of microbial transglutaminase of a newly isolated enzyme from Streptomyces spp. J. Macedo ∗ , H. Sato UNICAMP, Campinas, Brazil
A new microbial transglutaminase (MTGase or MTG, EC 2.3.2.13) from a Streptomyces sp. strain isolated from Brazilian soil samples had its production investigated, and was characterized in crude and purified forms. This work provides information about the production of this new enzyme, and its characterization, comparing its characteristics with the well-known commercial transglutaminase from Ajinomoto Co. Inc. (Activa® TG-BP). The Streptomyces sp. CBMAI 837 was chosen among a total of 200 actinomycetes strains isolated from Brazilian soil samples. To optimize the MTGase activity, modifications on the usual media compounds described for enzyme production were tested. The strategy employed to optimize the concentration of the key components were (1) screening experiment for the best carbon and nitrogen sources, (2) fractional factorial design (FFD) to elucidate the key ingredients and (3) central composite design (CCD). The experimental results were fitted with a second-order polynomial model at 95% level (P < 0.05). The optimized fermentation medium consisted of 2% soybean flour, 2% potato starch, 0.2% glucose, 2% peptone 0.2% KH2 PO4 and 0.1% MgSO4 ·7H2 O. Under the proposed optimized conditions, the model predicted the MTGase activity of 1.37 U/mL, very closely matching experimental activities of 1.4 U/mL. The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0—6.5 pH range and at 35—40◦ C. The results for the commercial enzyme were the same. A second maximum of activity was observed at pH 10.0 with both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been reported in the literature previously. The fact that this second maximum of activity does not appear on the purified form of the enzyme may suggest the presence of two isoenzymes on the crude extract. All of the enzymes tested were stable over the pH range from 4.5 to 8.0 and up to 45◦ C. The decline in activity of the commercial transglutaminase above 45◦ C and pH 8.0 was more gradual. The activities of all the MTG samples were independent of Ca2+ concentration, but they were elevated in the presence of K+ , Ba2+ , and Co2+ and inhibited by Cu2+ and Hg2+ , which suggests the presence of a thiol group in the MTG’s active site. The purified www.elsevier.com/locate/nbt S139
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
enzyme presented a Km of 6.37 mM and a Vmax of 1.7 U/mL, while the crude enzyme demonstrated a Km of 6.52 mM and a Vmax of 1.35 U/mL. doi:10.1016/j.nbt.2009.06.460
2.3.080 Optimization of lipase biocatalytic through sol—gel immobilization
performances
F. Peter ∗ , C. Zarcula, R. Croitoru, L. Corici University “Politehnica” of Timisoara, Timisoara, Romania
2.3.078 Asymmetric reduction of -carbonyl phenylpropionate by undifferentiated cells of white turnip in phosphate buffer/organic solvent Z. Ou ∗ , G. Yang Pharmaceuticals College, Zhejiang University of Technology, Hang Zhou, Zhejiang, China
(S)-(−)-Beta-hydroxy-benzenepropanoic acid ethyl ester was synthesized by asymmetric reduction of -carbonyl phenylpropionate with undifferentiated cells of white turnip in phosphate buffer/organic solvent. The yield increased with the LogPoct of organic solvent increase. The phosphate buffer (0.2 mol/L, pH 7.0)/dodecane was selected as optimum medium for reduction. The optimal content of dodecane in medium is 10% (v). The yield decreased with initial substrate concentration increase. The inhibition of substrate on reduction was not observed at 3.6—8.1 mmol/L of initial substrate concentration. Addition of 3.6 mmol/L substrate, the yield and the enantiometric excess of (S)(−)-Beta-hydroxy-benze-ne-propanoic acid ethyl ester reach 85.6% and 100% with 1% allyl bromide as inhibitor. doi:10.1016/j.nbt.2009.06.461
2.3.079 Operation and performance of analytical packed bed reactor with an immobilized enzyme on stainless-steel S.K. Arya 1,∗ , S.K. Srivastava 2 1
2
Department of Biotechnology, U.I.E.T., Panjab University, Chandigarh, India School of Biochemical Engineering, I.T. B.H.U., Varanasi, India
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) was immobilized with sodium alginate on stainless-steel mesh in packed bed bioreactor. The temperature was maintained at 60◦ C. Maximum product concentration was attained 40 mg -CD ml−1 at a dilution rate of 0.02 min−1 and maximum productivity attained 3.7 mg/ml/min−1 at a dilution rate of 0.08 min−1 . The Km,app value was 23.4 and 20.2 mg starch/ml at flow rate 2.0 and 10.0 ml min−1 , respectively, when extrapolate to near diffusion free condition. Reactor capacity 42 and 40 mg starch/min−1 at flow rate 2.0 and 10.0 ml min−1 , respectively. About 10% of decrease in productivity was observed after 10 days of operation. doi:10.1016/j.nbt.2009.06.462
Biocatalysts with improved functionalities are required for both academic research and industry, as high enzyme activity and enantioselectivity are key issues for large-scale applications. Lipases are well recognized as versatile catalysts in organic syntheses as they are able to accept a broad range of unnatural substrates and act in nonconventional systems. Immobilization allows enzymes to perform as any heterogeneous catalyst in various reaction media, recommending them for multiple reuse and application in continuous processes. The sol—gel immobilization techniques were developed mainly over the past decade, based on the building of a porous gel network around the enzyme molecule to yield an inert silica matrix with controlled porosity, increased mechanical resistance, and improved operational stability. Our research was focused on immobilization of commercial lipases from Pseudomonas fluorescens, Burkholderia cepacia and Candida antarctica, using methods based on sol—gel entrapment and sol—gel entrapment combined with adsorption. Tetramethoxysilane or tetraethoxysilane, jointly with silanes holding an alkyl or aryl nonhydrolizable group, have been used as precursors and ionic liquids were tested as immobilization additives. Optimization of the enzyme catalytic function was made for acylation reactions of aliphatic secondary alcohols, considering both activity and enantioselectivity. Although immobilization is commonly considered a method to improve the stability of enzymes, we obtained several preparates with better activity and enantiomeric ratio values compared to the native lipase, but in kinetic resolutions very high activity values were obviously associated to lower enantioselectivities. Ionic liquids as immobilization additives proved a positive effect on the activity of sol—gel entrapped lipases. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy, as well as FT-IR analysis demonstrated that the ionic liquids were partially incorporated in the structure of the formed xerogel and contributed to a better microenvironment for enzyme action. As reaction media, ionic liquids containing alkyl-immidazolium cationic moieties, particularly [Bmim]PF6, led to higher activities than the most efficient organic solvent (hexane). Sol—gel entrapment with ionic liquid [Omim]BF4 as immobilization additive was the optimal method to obtain highly active preparates, the relative total activity of P. fluorescens lipase increasing up to 3.3-fold next to immobilization. As regards enantioselectivity, ionic liquids were not so efficient reaction media as the best organic solvent, acetone, which gave enantiomeric ratio values higher than 100 for the acylation of 2-hexanol, compared to 15 in hexane and 44 in the best ionic liquid. Nevertheless, the optimization of biocatalytic function of sol—gel immobilized lipases should be tailored for every substrate and application. doi:10.1016/j.nbt.2009.06.463
S140
www.elsevier.com/locate/nbt
water. It is also possible to incorporate other materials, such as, metal particles in the Ion Jelly during its production. Ion Jelly® materials provide compatible microenvironments for enzymes such as oxi-reductases (e.g. HRP, glucose oxidase, among others) recently used in biosensors which showed excellent activity, and high operational and storage stability compared with other successful immobilization methods for the same group of enzymes, making it possible to develop colorimetric/electrochemical biosensors. The direct electron transfer between the immobilized proteins and enzymes and the modified electrodes were already successfully attained and proved using cyclic voltammetry. Besides the immobilization of large molecules, Ion Jelly® has proved to be able to incorporate smaller molecules and to release it slowly, namely with the application of potential variations, as tested with ferrocyanide. The interesting features of the material, together with the low cost of production and incorporation of possible catalysts by replacing small amount of gelatin, one of the constituents of Ion Jelly materials, make it suitable also in the field of new biomaterials for biosensors, either colorimetric and/or electrochemical detection and biofuel cells. doi:10.1016/j.nbt.2009.06.458
2.3.076 Effect of enzyme inactivation on kinetic parameters of immobilized penicillin G acylase in the hydrolysis of penicillin G J. Holtheuer , O. Romero, P. Valencia, A. Illanes, L. Wilson School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2147, Valparaíso, Chile
Enzyme inactivation is a major concern in biocatalytic reactions conducted under harsh process conditions. Kinetic parameters of enzyme biocatalysts are usually considered constant throughout reactor operation, which may not be the case if significant enzyme inactivation has occurred. This may be the case in the synthesis of -lactam antibiotics with penicillin G acylase (PGA), where the use of organic cosolvents is required to displace the equilibrium of the reaction towards synthesis, if the reaction is carried out under thermodynamic control, or to depress the competing hydrolytic reactions, in the case of kinetic control. The present study refers to the effect of partial inactivation of immobilized PGA in organic cosolvents on the kinetic parameters of the biocatalyst. A commercial preparation of E. coli PGA immobilized in methacrylate polymer (Dalenz—PGA) was a kind gift from Dalas Biotech (India) and used throughout this study. Kinetic parameters were determined from initial rate data of penicillin G hydrolysis at different substrate (penicillin G) and products concentrations (6-aminopenicillanic acid, 6APA, and phenyl acetic acid, PAA), determining the corresponding inhibition mechanisms. Inactivation was done at 50 and 85% (v/v) of dioxane down to a residual activity of 10 ± 2% and the kinetic parameters were determined for such inactivated biocatalysts and compared with those of the fresh enzyme. Michaelis constant (Km ) and inhibition constant by PAA (KPAA ) were reduced to 25% of their initial values after inactivation
ABSTRACTS
in dioxane (no significant differences were observed with respect to the dioxane concentration used), while inhibition constant by 6APA (K6APA ) was reduced to 10. Diffusional restrictions of the biocatalysts at reaction conditions cannot explain the magnitude of the differences observed in kinetic parameters that are to a great extent because of structural modifications of the enzyme during inactivation. Significant variation in kinetic parameters occurred as a consequence of enzyme inactivation that should be taken into account when modeling enzyme reactor performance, an aspect to which not much attention has been paid up to now. doi:10.1016/j.nbt.2009.06.459
2.3.077 Production and biochemical characterization of microbial transglutaminase of a newly isolated enzyme from Streptomyces spp. J. Macedo ∗ , H. Sato UNICAMP, Campinas, Brazil
A new microbial transglutaminase (MTGase or MTG, EC 2.3.2.13) from a Streptomyces sp. strain isolated from Brazilian soil samples had its production investigated, and was characterized in crude and purified forms. This work provides information about the production of this new enzyme, and its characterization, comparing its characteristics with the well-known commercial transglutaminase from Ajinomoto Co. Inc. (Activa® TG-BP). The Streptomyces sp. CBMAI 837 was chosen among a total of 200 actinomycetes strains isolated from Brazilian soil samples. To optimize the MTGase activity, modifications on the usual media compounds described for enzyme production were tested. The strategy employed to optimize the concentration of the key components were (1) screening experiment for the best carbon and nitrogen sources, (2) fractional factorial design (FFD) to elucidate the key ingredients and (3) central composite design (CCD). The experimental results were fitted with a second-order polynomial model at 95% level (P < 0.05). The optimized fermentation medium consisted of 2% soybean flour, 2% potato starch, 0.2% glucose, 2% peptone 0.2% KH2 PO4 and 0.1% MgSO4 ·7H2 O. Under the proposed optimized conditions, the model predicted the MTGase activity of 1.37 U/mL, very closely matching experimental activities of 1.4 U/mL. The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0—6.5 pH range and at 35—40◦ C. The results for the commercial enzyme were the same. A second maximum of activity was observed at pH 10.0 with both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been reported in the literature previously. The fact that this second maximum of activity does not appear on the purified form of the enzyme may suggest the presence of two isoenzymes on the crude extract. All of the enzymes tested were stable over the pH range from 4.5 to 8.0 and up to 45◦ C. The decline in activity of the commercial transglutaminase above 45◦ C and pH 8.0 was more gradual. The activities of all the MTG samples were independent of Ca2+ concentration, but they were elevated in the presence of K+ , Ba2+ , and Co2+ and inhibited by Cu2+ and Hg2+ , which suggests the presence of a thiol group in the MTG’s active site. The purified www.elsevier.com/locate/nbt S139
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
enzyme presented a Km of 6.37 mM and a Vmax of 1.7 U/mL, while the crude enzyme demonstrated a Km of 6.52 mM and a Vmax of 1.35 U/mL. doi:10.1016/j.nbt.2009.06.460
2.3.080 Optimization of lipase biocatalytic through sol—gel immobilization
performances
F. Peter ∗ , C. Zarcula, R. Croitoru, L. Corici University “Politehnica” of Timisoara, Timisoara, Romania
2.3.078 Asymmetric reduction of -carbonyl phenylpropionate by undifferentiated cells of white turnip in phosphate buffer/organic solvent Z. Ou ∗ , G. Yang Pharmaceuticals College, Zhejiang University of Technology, Hang Zhou, Zhejiang, China
(S)-(−)-Beta-hydroxy-benzenepropanoic acid ethyl ester was synthesized by asymmetric reduction of -carbonyl phenylpropionate with undifferentiated cells of white turnip in phosphate buffer/organic solvent. The yield increased with the LogPoct of organic solvent increase. The phosphate buffer (0.2 mol/L, pH 7.0)/dodecane was selected as optimum medium for reduction. The optimal content of dodecane in medium is 10% (v). The yield decreased with initial substrate concentration increase. The inhibition of substrate on reduction was not observed at 3.6—8.1 mmol/L of initial substrate concentration. Addition of 3.6 mmol/L substrate, the yield and the enantiometric excess of (S)(−)-Beta-hydroxy-benze-ne-propanoic acid ethyl ester reach 85.6% and 100% with 1% allyl bromide as inhibitor. doi:10.1016/j.nbt.2009.06.461
2.3.079 Operation and performance of analytical packed bed reactor with an immobilized enzyme on stainless-steel S.K. Arya 1,∗ , S.K. Srivastava 2 1
2
Department of Biotechnology, U.I.E.T., Panjab University, Chandigarh, India School of Biochemical Engineering, I.T. B.H.U., Varanasi, India
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) was immobilized with sodium alginate on stainless-steel mesh in packed bed bioreactor. The temperature was maintained at 60◦ C. Maximum product concentration was attained 40 mg -CD ml−1 at a dilution rate of 0.02 min−1 and maximum productivity attained 3.7 mg/ml/min−1 at a dilution rate of 0.08 min−1 . The Km,app value was 23.4 and 20.2 mg starch/ml at flow rate 2.0 and 10.0 ml min−1 , respectively, when extrapolate to near diffusion free condition. Reactor capacity 42 and 40 mg starch/min−1 at flow rate 2.0 and 10.0 ml min−1 , respectively. About 10% of decrease in productivity was observed after 10 days of operation. doi:10.1016/j.nbt.2009.06.462
Biocatalysts with improved functionalities are required for both academic research and industry, as high enzyme activity and enantioselectivity are key issues for large-scale applications. Lipases are well recognized as versatile catalysts in organic syntheses as they are able to accept a broad range of unnatural substrates and act in nonconventional systems. Immobilization allows enzymes to perform as any heterogeneous catalyst in various reaction media, recommending them for multiple reuse and application in continuous processes. The sol—gel immobilization techniques were developed mainly over the past decade, based on the building of a porous gel network around the enzyme molecule to yield an inert silica matrix with controlled porosity, increased mechanical resistance, and improved operational stability. Our research was focused on immobilization of commercial lipases from Pseudomonas fluorescens, Burkholderia cepacia and Candida antarctica, using methods based on sol—gel entrapment and sol—gel entrapment combined with adsorption. Tetramethoxysilane or tetraethoxysilane, jointly with silanes holding an alkyl or aryl nonhydrolizable group, have been used as precursors and ionic liquids were tested as immobilization additives. Optimization of the enzyme catalytic function was made for acylation reactions of aliphatic secondary alcohols, considering both activity and enantioselectivity. Although immobilization is commonly considered a method to improve the stability of enzymes, we obtained several preparates with better activity and enantiomeric ratio values compared to the native lipase, but in kinetic resolutions very high activity values were obviously associated to lower enantioselectivities. Ionic liquids as immobilization additives proved a positive effect on the activity of sol—gel entrapped lipases. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy, as well as FT-IR analysis demonstrated that the ionic liquids were partially incorporated in the structure of the formed xerogel and contributed to a better microenvironment for enzyme action. As reaction media, ionic liquids containing alkyl-immidazolium cationic moieties, particularly [Bmim]PF6, led to higher activities than the most efficient organic solvent (hexane). Sol—gel entrapment with ionic liquid [Omim]BF4 as immobilization additive was the optimal method to obtain highly active preparates, the relative total activity of P. fluorescens lipase increasing up to 3.3-fold next to immobilization. As regards enantioselectivity, ionic liquids were not so efficient reaction media as the best organic solvent, acetone, which gave enantiomeric ratio values higher than 100 for the acylation of 2-hexanol, compared to 15 in hexane and 44 in the best ionic liquid. Nevertheless, the optimization of biocatalytic function of sol—gel immobilized lipases should be tailored for every substrate and application. doi:10.1016/j.nbt.2009.06.463
S140
www.elsevier.com/locate/nbt