- Email: [email protected]
Protein engineering applications on Candida methylica formate dehydrogenase to elucidate folding mechanisms and to increase the thermostability
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
S81
taining the apramycin resistance gene as selectable marker. The gene disruption cassette was amplified from pIJ773 by PCR and introduced into E. coli BW25113/pIJ790 containing plasmid on which ppk gene is cloned. The isolated mutant plasmid was verified by restriction analysis and was introduced into nonmethylating E. coli ET12567. Then the mutant plasmid was transferred to B. thuringiensis by electroporation. The plasmid can only be maintained if it integrates by homologous recombination since it cannot replicate autonomously in Bacillus. At the end double cross-over transformants were screened for their antibiotic resistance.
Michaelis–Menten kinetic models. In the present study the reaction kinetics of sesame cake protein hydrolysis by Alcalase was investigated to determine decay effects for Alcalase. The reactions were carried out for 5 min in 0.1 L of aqueous solutions containing 10, 15, 20, 25 and 30 g protein/L at various temperature and pH values. For each experimental run, the kinetic parameters were estimated from Lineweaver–Burk plots. As a result of the experimental studies, Vmax values increased with temperature, while the Km values decreased. On the other hand, both of Vmax and Km values increased with the pH.
doi:10.1016/j.copbio.2011.05.241
doi:10.1016/j.copbio.2011.05.243
H5
H7
Evaluation of effects of technetium (99m Tc) pertechnetate on rat
Methanobrevibacter smithii carbamoyl phosphate synthetase, a missing link and a putative target for obesity prevention
Serdal Ogut, Mumin Polat, Gokhan Cesur, Dilek Karatopuk Ulusoy, Mustafa Yildiz, Fatih Gultekin
Elena Popa, Andreea Rusu, Cristina Purcarea
Practice and Research Hospital, Suleyman Demirel University, Isparta, Turkey
Institute of Biology Bucharest, Department of Microbiology, Bucharest, Romania
E-mail address: [email protected] (M. Polat)
E-mail address: [email protected] (C. Purcarea)
Technetium (99m Tc) pertechnetate is used for thyroid sintigraphy and thyroid uptake studies with success in radiology. In this study, we aimed to investigate the effects of 99m Tc pertechnetate on liver via using biochemical methods. Twenty female Wistar albino rats were included in this study. Animals were separated into two groups as follows: Controls (Group I), 37 MBq IV 99m Tc (Group II). At the end of the study, rats were sacrificed under ketaminexylasine anesthesia blood samples were collected. In Group II, there was 99m Tc pertechnetate induced liver damage, when compared with the control group. Biochemical analysis, there was a statistically significant increase in AST, ALT, GGT, LDH levels which are important markers of liver damage. Biochemical analysis, there was a statistically significant increase in LDH, AST, ALT, GGT levels which are important markers of liver damage. Consequently, we can say that 99m Tc pertechnetate causes damage in liver tissue.
Effect of temperature and pH on kinetics of sesame cake hydrolysis
The contribution of Methanobrevibacter smithii, the dominant archaeon in human gut, to improve our digestive system appears to involve ammonia assimilation. This study aims to characterize the structure and function of M. smithii carbamoyl phosphate synthetase (CPS), a key enzyme of ammonia metabolism, and to determine a mechanism of host obesity control by regulating the presence of the methanogen in the intestinal microbiota. M. smithii contains two CPSs that catalyze the precursor of the arginine and pyrimidines biosynthetic pathways, encoded by one carA gene (GLN subunit) and two carB genes (SYN subunits) of very different size (367-residue SYN1 and 1058-residue SYN2) but with conserved active sites. Remarkably, SYN1 appears to be the smallest CPS subunit identified so far. These CPS genes were cloned and expressed in E. coli and the recombinant proteins were purified by affinity chromatography. The CPS subunits exhibit glutaminase (GLN) and ammonium-dependent CPS activity (SYN), respectively. Kinetic parameters and regulatory response of the M. smithii small and large reconstituted CPSs indicate a particular type of carbamoyl phosphate synthesis, possibly specific for enteric methanogens. Structural–functional characteristics of M. smithii CPSs reveal a hypothetical missing link in the evolution of CPSases, and indicate a putative obesity controlling target.
Elcin Demirhan, Dilek Kilic Apar, Belma Ozbek
doi:10.1016/j.copbio.2011.05.244
Chemical Engineering Department, Yildiz Technical University, Istanbul, Turkey
H8
doi:10.1016/j.copbio.2011.05.242
H6
E-mail address: [email protected] (E. Demirhan) Plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. Protein hydrolysis has been shown to produce interesting effects on their technological properties such as solubility, foaming, emulsifying and gelation. It is possible to improve functional properties of proteins by chemical or enzymatic modifications. The use of enzymes provides milder process condition and allows for a selective hydrolysis of protein. Sesame protein may become an important source of high quality food protein for supplementing other plant protein, such as peanut, soybeans and other legumes, in tropical diets because of its high methionine and tryptophan content. In general, the reaction rates of enzymatic hydrolysis characterized according to
Protein engineering applications on Candida methylica formate dehydrogenase to elucidate folding mechanisms and to increase the thermostability Emel Ordu 1 , Nevin Gul Karaguler 2 1
Department of Biology, Faculty of Science and Letters, Yildiz Technical University, Istanbul, Turkey 2 Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey E-mail address: [email protected] (E. Ordu) NAD+-dependent formate dehydrogenase is used in industrial redox chemistry to regenerate NADH from NAD+. Despite many advantages of FDH, lack of thermostability is the problem of
S82
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
enzyme. In this study, initially, an efficient method was described to improve the purification of FDH from yeast Candida methylica (cmFDH). It allowed us to easily purify the constructed thermostabile mutants. The second section describes the folding mechanism and stability of dimeric native cmFDH. Equilibrium denaturation data yielded a dissociation constant of about 10−13 m. Findings showed that native cmFDH unfolds by two state single transition model in equilibrium. The kinetics of refolding and unfolding reactions revealed that the overall process comprises two steps. The rate of dissociation of the dimeric state in physiological conditions is extremely slow. Finally, rational design approach was applied to increase the thermostability of cmFDH. The thermodynamic and kinetic results suggested that except relatively improved mutants, four mutations increased the melting temperature between 2◦ C and 6◦ C greater than that of the native cmFDH, while the folding and unfolding patterns of native cmFDH was not altered. doi:10.1016/j.copbio.2011.05.245
H9 Immobilization of cellulase and xylanase on different supports Sheila Romo Sánchez 1 , Héctor Luis Ramirez Perez 2 , María Arévalo Villena 1 , Juan Úbeda Iranzo 1 1
Food Science and Technology, Castilla La Mancha University, Ciudad Real, Spain 2 Center for Enzyme Technology, Matanzas University, Matanzas, Cuba E-mail address: [email protected] (S.R. Sánchez) Many industries use enzymes during elaboration processes that have to be eliminated at the end. The recovery and reuse of them would suppose an important decrease of production cost. Therefore, our study aims at immobilizating cellulase and xylanase on different supports for their use in industry. The native enzymes were immobilized on different supports (chitosan, alginate, carboximetilcellulase (CMC)) and pectin, all of them with chitin, by using different techniques: adsorption and covalent bonding by activation with glutaraldehyde. The optimum conditions for immobilization were studied: protein concentration, time of contact and pH conditions. The enzymatic activity was determined by reaction between 100 l of enzyme solution to 400 l of 1% (w/v) substrate during 30 min that was stopped by adding 3,5dinitrosalicylic acid. Liberated reducing sugars were determined colorimetrically after boiling solutions for 10 min. Cellulase showed the best results on chitosan–chitin using the covalent bond with glutaraldehyde (0.125%). Cellulases immobilized by covalent bond with chitosan–chitin could be satisfactory for using in biotechnological process. doi:10.1016/j.copbio.2011.05.246
H10 Characterization of chitinase genes orginated from Serratia marcescens and their additive insecticidal activities to Bacillus thuringiensis strains Arzu Ozgen, Remziye Nalcacioglu, Kazim Sezen, Zihni Demirbag Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey E-mail address: [email protected] (Z. Demirbag) Characterizing the chitinase genes of Serratia marcescens and improving the insecticidal potential of Bacillus thuringiensis strains by cloning the chitinase genes. Chitinase B and chitinase C genes of S. marcescens isolated from Xyleborus dispar, amplified with degenerate primers by polymerase chain reaction. Nucleotide sequences of the genes and their deduced amino acid sequences were determined and compared with other chitinase genes. The genes were cloned into the pHY300PLK shuttle vector and the recombinant plasmids were introduced into E. coli DH10 and various strains of Bacillus thuringiensis (Bt). The stability of plasmids was examined for engineered E. coli DH10 and Bt strains. The insecticidal activity of engineered Bt strains were studied against larvae of Galleria mellonella (Lepidoptera) and Spodoptera littoralis. Chitinase B and chitinase C genes of S. marcescens have 96% homology with S. marcescens (BJL200) chiB gene and 96% homology with S. marcescens chiC (AJ630582.1), respectively. Recombinant plasmids showed varied decreasing stabilities in E. coli DH10 and Bt strains. Engineered Bt strains showed higher insecticidal activities than parental Bt strains and Serratia marcescens. The engineered Bt has potential to be utilized as a biocontrol agent in agriculture against some pests. doi:10.1016/j.copbio.2011.05.247
H11 Cloning and expression of keratinase gene from Bacillus sp. MKR1 Fatemeh Dabbagh, Maryam Shahbazi, Abdollah Ghasemian, Sara Rasoul Amini, Younes Ghasemi Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran E-mail address: [email protected] (F. Dabbagh) Kertinases constitute a distinct class of proteolytic enzymes capable of degrading keratin. Bioconversion of keratinous wastes into valuable products make them important enzymes in biotechnological approaches. Hence, the overproduction of keratinase through gene cloning and developing the appropriate inducible and applicable systems are important. In this study, we report the cloning, functional expression and characterization of keratinase from a newly isolated Bacillus sp. The keratinolytic strain Bacillus sp. MKR1 was used as a source of keratinase. The constructed recombinant pET15b/keratinase was introduced in Escherichia coli. In addition to enzymatic characterization of recombinant keratinase, the proteolytic and keratinolytic activity was assessed. Using E. coli transformed with recombinant pET/keratinase, the gene was expressed. The recombinant keratinase is active at a wide range of pH and temperature with an optimum occurring at pH 8 and 70◦ C. This study provides evidence that keratinase can be actively expressed in E. coli. To achieve a higher
S81
taining the apramycin resistance gene as selectable marker. The gene disruption cassette was amplified from pIJ773 by PCR and introduced into E. coli BW25113/pIJ790 containing plasmid on which ppk gene is cloned. The isolated mutant plasmid was verified by restriction analysis and was introduced into nonmethylating E. coli ET12567. Then the mutant plasmid was transferred to B. thuringiensis by electroporation. The plasmid can only be maintained if it integrates by homologous recombination since it cannot replicate autonomously in Bacillus. At the end double cross-over transformants were screened for their antibiotic resistance.
Michaelis–Menten kinetic models. In the present study the reaction kinetics of sesame cake protein hydrolysis by Alcalase was investigated to determine decay effects for Alcalase. The reactions were carried out for 5 min in 0.1 L of aqueous solutions containing 10, 15, 20, 25 and 30 g protein/L at various temperature and pH values. For each experimental run, the kinetic parameters were estimated from Lineweaver–Burk plots. As a result of the experimental studies, Vmax values increased with temperature, while the Km values decreased. On the other hand, both of Vmax and Km values increased with the pH.
doi:10.1016/j.copbio.2011.05.241
doi:10.1016/j.copbio.2011.05.243
H5
H7
Evaluation of effects of technetium (99m Tc) pertechnetate on rat
Methanobrevibacter smithii carbamoyl phosphate synthetase, a missing link and a putative target for obesity prevention
Serdal Ogut, Mumin Polat, Gokhan Cesur, Dilek Karatopuk Ulusoy, Mustafa Yildiz, Fatih Gultekin
Elena Popa, Andreea Rusu, Cristina Purcarea
Practice and Research Hospital, Suleyman Demirel University, Isparta, Turkey
Institute of Biology Bucharest, Department of Microbiology, Bucharest, Romania
E-mail address: [email protected] (M. Polat)
E-mail address: [email protected] (C. Purcarea)
Technetium (99m Tc) pertechnetate is used for thyroid sintigraphy and thyroid uptake studies with success in radiology. In this study, we aimed to investigate the effects of 99m Tc pertechnetate on liver via using biochemical methods. Twenty female Wistar albino rats were included in this study. Animals were separated into two groups as follows: Controls (Group I), 37 MBq IV 99m Tc (Group II). At the end of the study, rats were sacrificed under ketaminexylasine anesthesia blood samples were collected. In Group II, there was 99m Tc pertechnetate induced liver damage, when compared with the control group. Biochemical analysis, there was a statistically significant increase in AST, ALT, GGT, LDH levels which are important markers of liver damage. Biochemical analysis, there was a statistically significant increase in LDH, AST, ALT, GGT levels which are important markers of liver damage. Consequently, we can say that 99m Tc pertechnetate causes damage in liver tissue.
Effect of temperature and pH on kinetics of sesame cake hydrolysis
The contribution of Methanobrevibacter smithii, the dominant archaeon in human gut, to improve our digestive system appears to involve ammonia assimilation. This study aims to characterize the structure and function of M. smithii carbamoyl phosphate synthetase (CPS), a key enzyme of ammonia metabolism, and to determine a mechanism of host obesity control by regulating the presence of the methanogen in the intestinal microbiota. M. smithii contains two CPSs that catalyze the precursor of the arginine and pyrimidines biosynthetic pathways, encoded by one carA gene (GLN subunit) and two carB genes (SYN subunits) of very different size (367-residue SYN1 and 1058-residue SYN2) but with conserved active sites. Remarkably, SYN1 appears to be the smallest CPS subunit identified so far. These CPS genes were cloned and expressed in E. coli and the recombinant proteins were purified by affinity chromatography. The CPS subunits exhibit glutaminase (GLN) and ammonium-dependent CPS activity (SYN), respectively. Kinetic parameters and regulatory response of the M. smithii small and large reconstituted CPSs indicate a particular type of carbamoyl phosphate synthesis, possibly specific for enteric methanogens. Structural–functional characteristics of M. smithii CPSs reveal a hypothetical missing link in the evolution of CPSases, and indicate a putative obesity controlling target.
Elcin Demirhan, Dilek Kilic Apar, Belma Ozbek
doi:10.1016/j.copbio.2011.05.244
Chemical Engineering Department, Yildiz Technical University, Istanbul, Turkey
H8
doi:10.1016/j.copbio.2011.05.242
H6
E-mail address: [email protected] (E. Demirhan) Plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. Protein hydrolysis has been shown to produce interesting effects on their technological properties such as solubility, foaming, emulsifying and gelation. It is possible to improve functional properties of proteins by chemical or enzymatic modifications. The use of enzymes provides milder process condition and allows for a selective hydrolysis of protein. Sesame protein may become an important source of high quality food protein for supplementing other plant protein, such as peanut, soybeans and other legumes, in tropical diets because of its high methionine and tryptophan content. In general, the reaction rates of enzymatic hydrolysis characterized according to
Protein engineering applications on Candida methylica formate dehydrogenase to elucidate folding mechanisms and to increase the thermostability Emel Ordu 1 , Nevin Gul Karaguler 2 1
Department of Biology, Faculty of Science and Letters, Yildiz Technical University, Istanbul, Turkey 2 Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey E-mail address: [email protected] (E. Ordu) NAD+-dependent formate dehydrogenase is used in industrial redox chemistry to regenerate NADH from NAD+. Despite many advantages of FDH, lack of thermostability is the problem of
S82
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
enzyme. In this study, initially, an efficient method was described to improve the purification of FDH from yeast Candida methylica (cmFDH). It allowed us to easily purify the constructed thermostabile mutants. The second section describes the folding mechanism and stability of dimeric native cmFDH. Equilibrium denaturation data yielded a dissociation constant of about 10−13 m. Findings showed that native cmFDH unfolds by two state single transition model in equilibrium. The kinetics of refolding and unfolding reactions revealed that the overall process comprises two steps. The rate of dissociation of the dimeric state in physiological conditions is extremely slow. Finally, rational design approach was applied to increase the thermostability of cmFDH. The thermodynamic and kinetic results suggested that except relatively improved mutants, four mutations increased the melting temperature between 2◦ C and 6◦ C greater than that of the native cmFDH, while the folding and unfolding patterns of native cmFDH was not altered. doi:10.1016/j.copbio.2011.05.245
H9 Immobilization of cellulase and xylanase on different supports Sheila Romo Sánchez 1 , Héctor Luis Ramirez Perez 2 , María Arévalo Villena 1 , Juan Úbeda Iranzo 1 1
Food Science and Technology, Castilla La Mancha University, Ciudad Real, Spain 2 Center for Enzyme Technology, Matanzas University, Matanzas, Cuba E-mail address: [email protected] (S.R. Sánchez) Many industries use enzymes during elaboration processes that have to be eliminated at the end. The recovery and reuse of them would suppose an important decrease of production cost. Therefore, our study aims at immobilizating cellulase and xylanase on different supports for their use in industry. The native enzymes were immobilized on different supports (chitosan, alginate, carboximetilcellulase (CMC)) and pectin, all of them with chitin, by using different techniques: adsorption and covalent bonding by activation with glutaraldehyde. The optimum conditions for immobilization were studied: protein concentration, time of contact and pH conditions. The enzymatic activity was determined by reaction between 100 l of enzyme solution to 400 l of 1% (w/v) substrate during 30 min that was stopped by adding 3,5dinitrosalicylic acid. Liberated reducing sugars were determined colorimetrically after boiling solutions for 10 min. Cellulase showed the best results on chitosan–chitin using the covalent bond with glutaraldehyde (0.125%). Cellulases immobilized by covalent bond with chitosan–chitin could be satisfactory for using in biotechnological process. doi:10.1016/j.copbio.2011.05.246
H10 Characterization of chitinase genes orginated from Serratia marcescens and their additive insecticidal activities to Bacillus thuringiensis strains Arzu Ozgen, Remziye Nalcacioglu, Kazim Sezen, Zihni Demirbag Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey E-mail address: [email protected] (Z. Demirbag) Characterizing the chitinase genes of Serratia marcescens and improving the insecticidal potential of Bacillus thuringiensis strains by cloning the chitinase genes. Chitinase B and chitinase C genes of S. marcescens isolated from Xyleborus dispar, amplified with degenerate primers by polymerase chain reaction. Nucleotide sequences of the genes and their deduced amino acid sequences were determined and compared with other chitinase genes. The genes were cloned into the pHY300PLK shuttle vector and the recombinant plasmids were introduced into E. coli DH10 and various strains of Bacillus thuringiensis (Bt). The stability of plasmids was examined for engineered E. coli DH10 and Bt strains. The insecticidal activity of engineered Bt strains were studied against larvae of Galleria mellonella (Lepidoptera) and Spodoptera littoralis. Chitinase B and chitinase C genes of S. marcescens have 96% homology with S. marcescens (BJL200) chiB gene and 96% homology with S. marcescens chiC (AJ630582.1), respectively. Recombinant plasmids showed varied decreasing stabilities in E. coli DH10 and Bt strains. Engineered Bt strains showed higher insecticidal activities than parental Bt strains and Serratia marcescens. The engineered Bt has potential to be utilized as a biocontrol agent in agriculture against some pests. doi:10.1016/j.copbio.2011.05.247
H11 Cloning and expression of keratinase gene from Bacillus sp. MKR1 Fatemeh Dabbagh, Maryam Shahbazi, Abdollah Ghasemian, Sara Rasoul Amini, Younes Ghasemi Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran E-mail address: [email protected] (F. Dabbagh) Kertinases constitute a distinct class of proteolytic enzymes capable of degrading keratin. Bioconversion of keratinous wastes into valuable products make them important enzymes in biotechnological approaches. Hence, the overproduction of keratinase through gene cloning and developing the appropriate inducible and applicable systems are important. In this study, we report the cloning, functional expression and characterization of keratinase from a newly isolated Bacillus sp. The keratinolytic strain Bacillus sp. MKR1 was used as a source of keratinase. The constructed recombinant pET15b/keratinase was introduced in Escherichia coli. In addition to enzymatic characterization of recombinant keratinase, the proteolytic and keratinolytic activity was assessed. Using E. coli transformed with recombinant pET/keratinase, the gene was expressed. The recombinant keratinase is active at a wide range of pH and temperature with an optimum occurring at pH 8 and 70◦ C. This study provides evidence that keratinase can be actively expressed in E. coli. To achieve a higher