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Enzymatic synthesis and ways of further treatment of fructooligosaccharides and polymeric levan for prebiotic efficiency studies
S122
Abstracts / New Biotechnology 33S (2016) S1–S213
The prepared hydrolysate was fortified with ammonium chloride as nitrogen source. A central composite design was conducted for determining the effects of initial xylose concentration, added ammonium chloride concentration and shaking rate on maximum xylitol concentration. Experiments were carried out in water bath shakers using 300 mL cotton-plugged flasks containing 80 mL of fermentation medium. The effects of independent variables were defined by a quadratic model. According to the ANOVA results, linear effects of each variables were found to be significant for maximum xylitol concentration. In addition, interaction effects of all parameters, as well as quadratic effects of added ammonium chloride concentration and shaking rate were determined as significant. It was also demonstrated that fortified chestnut shell hydrolysate could be used for xylitol production by C. tropicalis. http://dx.doi.org/10.1016/j.nbt.2016.06.1145
P11-20 Kinetic modeling of the vitamin K2 production with Flavobacterium sp. cultured on glycerol Zhiming Zheng 1,∗ , Hefang Wu 2 , Han Wang 1 , Peng Wang 2 , Genhai Zhao 1 1 2
Hefei Institutes of Physical Science, China Chinese Academy of Sciences, China
Establishing a mathematical model is a premise to optimization of fermentation process control. However the kinetic study for Flavobacterium sp. as the potential bacteria for vitamin K2 production has never been considered. A logistic-based model of Flavobacterium sp. was developed to quantify growth kinetics with glycerol and the production of vitamin K2 by batch fermentation, and glycerol consumption were also developed. The vitamin K2 production of the original strain f1 and the mutant strain f2 were investigated. The results of both strains were successfully simulated using the developed kinetics model. The vitamin K2 fermentation with Flavobacterium sp. as the producer was typical part of growth coupling. The vitamin K2 accumulation combined growth associated and non-growth associated contributions, and the nongrowth associated part for vitamin K2 production contributed the most important influence. The resistant mutant (f2) was characterized by a higher growth rate (m = 0.110 vs. 0.0627 h−1 ) and more vitamin K2 production (f2 = 12.46 mg/L vs. 6.63 mg/L) compared to the f1. On the strength of characteristics of product formation and substrate utilization, a fed-batch operation strategy was adopted in order to obtain more vitamin K2 accumulation. The final production of vitamin K2 reached 13.5 mg/L in 216 h of fed-batch fermentation. http://dx.doi.org/10.1016/j.nbt.2016.06.1146
P11-21 Selection of new fungal strain producing feruloyl esterases for biotechnological applications Simona Varriale 1,∗ , Gabriella Cerullo 1 , Valeria Ventorino 1 , Olimpia Pepe 1 , Martin Meijer 2 , Ronald De Vries 2 , Vincenza Faraco 1 1 2
University of Naples “Federico II”, Italy CBS-KNAW Fungal Biodiversity Centre, The Netherlands
Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases secreted by a wide range of fungi and
bacteria. These enzymes have the ability to catalyze hydrolysis of the ester linkage of hydroxycinnamic acids and diferulates present in plant cell-walls, but also to synthesize a broad range of novel bioactive compounds for use in food, cosmetic and pharmaceutical industries. Our aim was to identify novel FAEs with non-conserved sequences. Therefore, fungi were isolated from lignocellulosic biomasses during biodegradation under natural conditions, and selected for the ability to synthesize enzymes with potentially synergistic action in lignocellulose conversion, including FAE enzymes. FAE activity was measured towards p-nitrophenyl-ferulate after liquid cultivation in the presence of de-starched wheat bran as sole carbon source. Moreover, the influence of ferulic acid as inducer of FAE activity production was explored, by adding it to the untreated wheat bran. Xylan was also tested as carbon source, whilst glucose was generally used as a negative control. The fungus with the highest FAE production in the presence of de-starched bran has been identified as Talaromyces sp. nov. (from section Helici) which has a great biotechnological potential because of its known ability to secrete numerous biopolymer degrading enzymes. Thus the fungus was further characterized. Acknowledgments: This research was supported by the OPTIBIOCAT Project (N. 613868). p-Nitrophenyl ferulate was provided by Dr. Alexander Piechot and Dr. Peter Jütten (Taros Chemicals GmbH & Co. KG). http://dx.doi.org/10.1016/j.nbt.2016.06.1147
Glycobiotechnology P12-1 Enzymatic synthesis and ways of further treatment of fructooligosaccharides and polymeric levan for prebiotic efficiency studies Karin Mardo 1,∗ , Triinu Visnapuu 2 , Anneli Aasamets 3 , Katrin Viigand 2 , Olesja Bondarenko 3 , Heiki Vija 2 , Kaarel Adamberg 3 , Signe Adamberg 4 , Tiina Alamäe 4 1
Tartu University, Estonia Institute of Molecular and Cell Biology, Estonia 3 University of Tartu, Estonia 4 National Institute of Chemical Physics and Biophysics, Estonia 2
Levan and levan-type fructooligosaccharides (FOS) are novel potential prebiotics that have gained little attention compared to inulin and inulin-type FOS. These two fructans types differ for the linkage between the fructose residues. We have cloned and expressed a highly active and robust bacterial levansucrase which produces from sucrose, raffinose and molasses polymeric levan and short-chain FOS. The enzyme is highly stable retaining its activity even at 37◦ over a year. So far we have elaborated a protocol for medium-scale production and purification of levan and levan-type FOS. FOS were treated by a yeast maltase that stripped its residues from the ends of the FOS chains, thus modifying the preparation. FOS and levan were used in fermentation experiment as a substrate for a gut commensal Bacteroides thetaiotaomicron and as a selective carbon source for human gut consortia. Cobalt from levan-coated nanoparticles was solubilized if the particles were incubated with B. thetaiotaomicron due to production of acidic fermentation products from levan. Levan-coated Co nanoparticles may be regarded as Co supply for gut bacteria needing this metal for the synthesis of Co-containing enzymes. Levan-type FOS can also be produced by enzymatic digestion of polymeric levan. For that, suitable commercial enzymes are not yet available. Some rumen bacteria and bacilli
Abstracts / New Biotechnology 33S (2016) S1–S213
possess endo-acting hydrolases of levan. We aim to search and use a suitable levan-splitting enzyme from some member of human gut microbiota in order to produce FOS from bacterial levan Acknowledgments: Financing from grants GLOMR9072 and GLTMR1050P and SLOMR12215T. http://dx.doi.org/10.1016/j.nbt.2016.06.1148
P12-2 Reaction temperature effect of enzymatically modified starches with amylosucrase from Deinococcus geothermalis Dong-Ho Seo 1,∗ , Hyun-Seok Kim 2 , Moo-Yeol Baik 3 , Choen-Seok Park 3 1
Korea Food Research Institute, South Korea Andong National University, South Korea 3 Kyung Hee University, South Korea
never before found in mammals. NOR is synthesized by the enzyme variant with p.Q211E substitution caused by c.631C>G mutation in A4GALT. The mutation broadens the enzyme specificity, rendering it able to synthesize NOR in addition to P1 and Pk . Using a recombinant catalytic domain of A4GALT obtained in High Five insect cells, we present the biochemical evidence that the consensus alpha1,4-galactosyltransferase may synthesize both, P1 and Pk antigens, while its p.Q211E variant is able to additionally synthesize NOR. A4GALT has two potential N-glycosylation sites. Analysis of the oligosaccharides released from the protein after PNGase F treatment revealed alpha1,6-fucosylated paucimannose structures, typical for insect glycoproteins, which shows that at least one of the potential N-glycosylation sites is occupied. In addition, removal of these glycans causes complete loss of A4GALT activity. To the best of our knowledge, this is the first case wherein N-glycans of a glycosyltransferase are crucial for its activity.
2
http://dx.doi.org/10.1016/j.nbt.2016.06.1150
Amylosucrae (ASae, EC 2.4.1.4) is not only synthesizes a-1,4glucans like amylose from sucrose as a sole substrate, but also can produce digestion-resistant starch (RS) using starches as an acceptor and sucrose as a donor. Recombinant ASase from Deinococcus geothermalis (DGAS) is highly thermostable among various ASases and is able to react at a wide range of temperature. Pre-gelatinised potato starch was treated with DGAS for 48 h at various temperatures (30, 40, and 50 ◦ C). DGAS-treated starches were characterized with respect to morphology, X-ray diffraction pattern, particle size distribution, thermal transition, solubility and swelling power, pasting properties, amylopectin branch-chain distribution, and RS content. After enzymatic reaction, RS contents increased by 23.5%, 30.9% and 14.1% from 4.2% of potato starch at 30, 40, and 50 ◦ C, respectively. The crystalline polymorph of DGAS-treated potato starches resulted in the B-type immediately after enzymatic reaction. As reaction temperature rise, solubility and swelling power of DGAS-treated potato starch has increased. Other properties of DGAS-treated potato starch were difference depending on reaction temperature. This study showed that reaction temperature affects ASase-treated starches properties.
P12-5
http://dx.doi.org/10.1016/j.nbt.2016.06.1149
P12-4 Human ␣1,4-galactosyltransferase (Gb3/CD77 synthase): Expression in insect cells, properties, role of glycans Katarzyna Szymczak ∗ , Radoslaw Kaczmarek, Maria Duk, Krzysztof Mikolajczyk, Agata Zerka, Anna Bereznicka, Mariusz Olczak, Marcin Czerwinski Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland
S123
Carbohydrate-binding modules interacting with polyethyleneterepthalate Joanna Weber 1,∗ , Duˇsan Petrovic´ 2 , Birgit Strodel 3 , Stephan Kolkenbrock 4 , Karl-Erich Jaeger 3 , Christian Leggewie 4 1 evocatal GmbH, Institute of Molecular Enzyme Technology, Germany 2 Heinrich-Heine-University Düsseldorf, Germany 3 Forschungszentrum Jülich, Germany 4 Institute of Complex Systems: Structural Biochemistry (ICS-6), Germany
Carbohydrate-binding modules (CBMs) are non-catalytic structures found in enzymes which interact with polysaccharides promoting the association of the enzyme with the substrate. Interestingly, some CBMs show binding affinity towards one of the most widely applied synthetic polymers, namely polyethyleneterepthalate (PET) [1]. In 2013, the worldwide production of PET averaged 56 million tons used primarily for fabrication of plastic bottles, films and textiles. Peptides can be used as auto-binding partners on PET surfaces thereby offering innovative application possibilities and smart solutions for the development of adhesives, the immobilization of enzymes or the functionalization of synthetic fibers. We have established a semi-quantitative method to detect CBMs on PET-films and have screened a selection of 8 CBMs for PET-binding revealing one CBM which possessed a high affinity towards PET. Molecular dynamics (MD) simulations were carried out to identify amino acids involved in binding. Based on this data, CBM variants were designed and analyzed for PET-binding resulting in the identification of engineered CBM variants with optimized binding affinity towards PET. Reference
Human alpha1,4-galactosyltransferase (A4GALT, Gb3/CD77 synthase, Pk synthase) synthesizes 2 or 3 carbohydrate antigens of the P1PK blood group system depending on whether position 211 of the polypeptide chain is occupied by Gln or Glu residue, respectively. P1, Pk and NOR antigens are carried by glycosphingolipids. P1 and Pk terminate with Gal(alpha1-4)Gal disaccharide and have long been thought to be synthesized by the consensus enzyme (encoded byA4GALT gene). The presence or absence of P1 antigen determines the P1 or P2 blood group, respectively. The rare NOR antigen (whose structure was solved in our laboratory) terminates with unusual Gal(alpha1-4)GalNAc disaccharide,
[1] Zhang Y, Wang L, Chen J, Wu J. Enhanced activity toward PET by site-directed mutagenesis of Thermobifida fusca cutinase-CBM fusion protein. Carbohyd Polym 2013;97:124–9.
http://dx.doi.org/10.1016/j.nbt.2016.06.1151
Abstracts / New Biotechnology 33S (2016) S1–S213
The prepared hydrolysate was fortified with ammonium chloride as nitrogen source. A central composite design was conducted for determining the effects of initial xylose concentration, added ammonium chloride concentration and shaking rate on maximum xylitol concentration. Experiments were carried out in water bath shakers using 300 mL cotton-plugged flasks containing 80 mL of fermentation medium. The effects of independent variables were defined by a quadratic model. According to the ANOVA results, linear effects of each variables were found to be significant for maximum xylitol concentration. In addition, interaction effects of all parameters, as well as quadratic effects of added ammonium chloride concentration and shaking rate were determined as significant. It was also demonstrated that fortified chestnut shell hydrolysate could be used for xylitol production by C. tropicalis. http://dx.doi.org/10.1016/j.nbt.2016.06.1145
P11-20 Kinetic modeling of the vitamin K2 production with Flavobacterium sp. cultured on glycerol Zhiming Zheng 1,∗ , Hefang Wu 2 , Han Wang 1 , Peng Wang 2 , Genhai Zhao 1 1 2
Hefei Institutes of Physical Science, China Chinese Academy of Sciences, China
Establishing a mathematical model is a premise to optimization of fermentation process control. However the kinetic study for Flavobacterium sp. as the potential bacteria for vitamin K2 production has never been considered. A logistic-based model of Flavobacterium sp. was developed to quantify growth kinetics with glycerol and the production of vitamin K2 by batch fermentation, and glycerol consumption were also developed. The vitamin K2 production of the original strain f1 and the mutant strain f2 were investigated. The results of both strains were successfully simulated using the developed kinetics model. The vitamin K2 fermentation with Flavobacterium sp. as the producer was typical part of growth coupling. The vitamin K2 accumulation combined growth associated and non-growth associated contributions, and the nongrowth associated part for vitamin K2 production contributed the most important influence. The resistant mutant (f2) was characterized by a higher growth rate (m = 0.110 vs. 0.0627 h−1 ) and more vitamin K2 production (f2 = 12.46 mg/L vs. 6.63 mg/L) compared to the f1. On the strength of characteristics of product formation and substrate utilization, a fed-batch operation strategy was adopted in order to obtain more vitamin K2 accumulation. The final production of vitamin K2 reached 13.5 mg/L in 216 h of fed-batch fermentation. http://dx.doi.org/10.1016/j.nbt.2016.06.1146
P11-21 Selection of new fungal strain producing feruloyl esterases for biotechnological applications Simona Varriale 1,∗ , Gabriella Cerullo 1 , Valeria Ventorino 1 , Olimpia Pepe 1 , Martin Meijer 2 , Ronald De Vries 2 , Vincenza Faraco 1 1 2
University of Naples “Federico II”, Italy CBS-KNAW Fungal Biodiversity Centre, The Netherlands
Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases secreted by a wide range of fungi and
bacteria. These enzymes have the ability to catalyze hydrolysis of the ester linkage of hydroxycinnamic acids and diferulates present in plant cell-walls, but also to synthesize a broad range of novel bioactive compounds for use in food, cosmetic and pharmaceutical industries. Our aim was to identify novel FAEs with non-conserved sequences. Therefore, fungi were isolated from lignocellulosic biomasses during biodegradation under natural conditions, and selected for the ability to synthesize enzymes with potentially synergistic action in lignocellulose conversion, including FAE enzymes. FAE activity was measured towards p-nitrophenyl-ferulate after liquid cultivation in the presence of de-starched wheat bran as sole carbon source. Moreover, the influence of ferulic acid as inducer of FAE activity production was explored, by adding it to the untreated wheat bran. Xylan was also tested as carbon source, whilst glucose was generally used as a negative control. The fungus with the highest FAE production in the presence of de-starched bran has been identified as Talaromyces sp. nov. (from section Helici) which has a great biotechnological potential because of its known ability to secrete numerous biopolymer degrading enzymes. Thus the fungus was further characterized. Acknowledgments: This research was supported by the OPTIBIOCAT Project (N. 613868). p-Nitrophenyl ferulate was provided by Dr. Alexander Piechot and Dr. Peter Jütten (Taros Chemicals GmbH & Co. KG). http://dx.doi.org/10.1016/j.nbt.2016.06.1147
Glycobiotechnology P12-1 Enzymatic synthesis and ways of further treatment of fructooligosaccharides and polymeric levan for prebiotic efficiency studies Karin Mardo 1,∗ , Triinu Visnapuu 2 , Anneli Aasamets 3 , Katrin Viigand 2 , Olesja Bondarenko 3 , Heiki Vija 2 , Kaarel Adamberg 3 , Signe Adamberg 4 , Tiina Alamäe 4 1
Tartu University, Estonia Institute of Molecular and Cell Biology, Estonia 3 University of Tartu, Estonia 4 National Institute of Chemical Physics and Biophysics, Estonia 2
Levan and levan-type fructooligosaccharides (FOS) are novel potential prebiotics that have gained little attention compared to inulin and inulin-type FOS. These two fructans types differ for the linkage between the fructose residues. We have cloned and expressed a highly active and robust bacterial levansucrase which produces from sucrose, raffinose and molasses polymeric levan and short-chain FOS. The enzyme is highly stable retaining its activity even at 37◦ over a year. So far we have elaborated a protocol for medium-scale production and purification of levan and levan-type FOS. FOS were treated by a yeast maltase that stripped its residues from the ends of the FOS chains, thus modifying the preparation. FOS and levan were used in fermentation experiment as a substrate for a gut commensal Bacteroides thetaiotaomicron and as a selective carbon source for human gut consortia. Cobalt from levan-coated nanoparticles was solubilized if the particles were incubated with B. thetaiotaomicron due to production of acidic fermentation products from levan. Levan-coated Co nanoparticles may be regarded as Co supply for gut bacteria needing this metal for the synthesis of Co-containing enzymes. Levan-type FOS can also be produced by enzymatic digestion of polymeric levan. For that, suitable commercial enzymes are not yet available. Some rumen bacteria and bacilli
Abstracts / New Biotechnology 33S (2016) S1–S213
possess endo-acting hydrolases of levan. We aim to search and use a suitable levan-splitting enzyme from some member of human gut microbiota in order to produce FOS from bacterial levan Acknowledgments: Financing from grants GLOMR9072 and GLTMR1050P and SLOMR12215T. http://dx.doi.org/10.1016/j.nbt.2016.06.1148
P12-2 Reaction temperature effect of enzymatically modified starches with amylosucrase from Deinococcus geothermalis Dong-Ho Seo 1,∗ , Hyun-Seok Kim 2 , Moo-Yeol Baik 3 , Choen-Seok Park 3 1
Korea Food Research Institute, South Korea Andong National University, South Korea 3 Kyung Hee University, South Korea
never before found in mammals. NOR is synthesized by the enzyme variant with p.Q211E substitution caused by c.631C>G mutation in A4GALT. The mutation broadens the enzyme specificity, rendering it able to synthesize NOR in addition to P1 and Pk . Using a recombinant catalytic domain of A4GALT obtained in High Five insect cells, we present the biochemical evidence that the consensus alpha1,4-galactosyltransferase may synthesize both, P1 and Pk antigens, while its p.Q211E variant is able to additionally synthesize NOR. A4GALT has two potential N-glycosylation sites. Analysis of the oligosaccharides released from the protein after PNGase F treatment revealed alpha1,6-fucosylated paucimannose structures, typical for insect glycoproteins, which shows that at least one of the potential N-glycosylation sites is occupied. In addition, removal of these glycans causes complete loss of A4GALT activity. To the best of our knowledge, this is the first case wherein N-glycans of a glycosyltransferase are crucial for its activity.
2
http://dx.doi.org/10.1016/j.nbt.2016.06.1150
Amylosucrae (ASae, EC 2.4.1.4) is not only synthesizes a-1,4glucans like amylose from sucrose as a sole substrate, but also can produce digestion-resistant starch (RS) using starches as an acceptor and sucrose as a donor. Recombinant ASase from Deinococcus geothermalis (DGAS) is highly thermostable among various ASases and is able to react at a wide range of temperature. Pre-gelatinised potato starch was treated with DGAS for 48 h at various temperatures (30, 40, and 50 ◦ C). DGAS-treated starches were characterized with respect to morphology, X-ray diffraction pattern, particle size distribution, thermal transition, solubility and swelling power, pasting properties, amylopectin branch-chain distribution, and RS content. After enzymatic reaction, RS contents increased by 23.5%, 30.9% and 14.1% from 4.2% of potato starch at 30, 40, and 50 ◦ C, respectively. The crystalline polymorph of DGAS-treated potato starches resulted in the B-type immediately after enzymatic reaction. As reaction temperature rise, solubility and swelling power of DGAS-treated potato starch has increased. Other properties of DGAS-treated potato starch were difference depending on reaction temperature. This study showed that reaction temperature affects ASase-treated starches properties.
P12-5
http://dx.doi.org/10.1016/j.nbt.2016.06.1149
P12-4 Human ␣1,4-galactosyltransferase (Gb3/CD77 synthase): Expression in insect cells, properties, role of glycans Katarzyna Szymczak ∗ , Radoslaw Kaczmarek, Maria Duk, Krzysztof Mikolajczyk, Agata Zerka, Anna Bereznicka, Mariusz Olczak, Marcin Czerwinski Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland
S123
Carbohydrate-binding modules interacting with polyethyleneterepthalate Joanna Weber 1,∗ , Duˇsan Petrovic´ 2 , Birgit Strodel 3 , Stephan Kolkenbrock 4 , Karl-Erich Jaeger 3 , Christian Leggewie 4 1 evocatal GmbH, Institute of Molecular Enzyme Technology, Germany 2 Heinrich-Heine-University Düsseldorf, Germany 3 Forschungszentrum Jülich, Germany 4 Institute of Complex Systems: Structural Biochemistry (ICS-6), Germany
Carbohydrate-binding modules (CBMs) are non-catalytic structures found in enzymes which interact with polysaccharides promoting the association of the enzyme with the substrate. Interestingly, some CBMs show binding affinity towards one of the most widely applied synthetic polymers, namely polyethyleneterepthalate (PET) [1]. In 2013, the worldwide production of PET averaged 56 million tons used primarily for fabrication of plastic bottles, films and textiles. Peptides can be used as auto-binding partners on PET surfaces thereby offering innovative application possibilities and smart solutions for the development of adhesives, the immobilization of enzymes or the functionalization of synthetic fibers. We have established a semi-quantitative method to detect CBMs on PET-films and have screened a selection of 8 CBMs for PET-binding revealing one CBM which possessed a high affinity towards PET. Molecular dynamics (MD) simulations were carried out to identify amino acids involved in binding. Based on this data, CBM variants were designed and analyzed for PET-binding resulting in the identification of engineered CBM variants with optimized binding affinity towards PET. Reference
Human alpha1,4-galactosyltransferase (A4GALT, Gb3/CD77 synthase, Pk synthase) synthesizes 2 or 3 carbohydrate antigens of the P1PK blood group system depending on whether position 211 of the polypeptide chain is occupied by Gln or Glu residue, respectively. P1, Pk and NOR antigens are carried by glycosphingolipids. P1 and Pk terminate with Gal(alpha1-4)Gal disaccharide and have long been thought to be synthesized by the consensus enzyme (encoded byA4GALT gene). The presence or absence of P1 antigen determines the P1 or P2 blood group, respectively. The rare NOR antigen (whose structure was solved in our laboratory) terminates with unusual Gal(alpha1-4)GalNAc disaccharide,
[1] Zhang Y, Wang L, Chen J, Wu J. Enhanced activity toward PET by site-directed mutagenesis of Thermobifida fusca cutinase-CBM fusion protein. Carbohyd Polym 2013;97:124–9.
http://dx.doi.org/10.1016/j.nbt.2016.06.1151