- Email: [email protected]
Phytase inactivation in the animal feed pelleting process
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
backs. Sugar cane processing sugar mills are highly integrated biorefineries producing sugar at lowest costs. Unfortunately, these sugar mills are limited to sugar cane growing regions which do not necessarily match with regions of chemical production and the corresponding chemical markets. Traditional wet mills are distributed all over the world as their raw material corn is a transportable good. As most of the produced glucose is used for human consumption, these plants are generally delivering food-quality which is not required for the fermentation industry. In addition, these facilities are highly capital-intensive which lead to large-scale facilities of 1.5 million tons corn crushing capacity to reduce the specific investment costs per ton of glucose produced. In general, this amount of glucose exceeds the demand of even a conglomerate of fermentation processes. Facing these obstacles BASF has developed a unique corn-based process for the production of low-cost fermentation-grade glucose, even at moderate scale facilities. This process has been realized at a pilot plant scale in conjunction with a fermentation and product recovery unit. Trials on the pilot plant revealed increases in titer and yield during fermentation compared to traditional sugar feedstocks. Compared to these feedstocks, the glucose solution is rich in nutrients. As a result, the amount of complex media components can be reduced, saving additional costs. The co-products generated in the process by refining of corn can be sold to the feed market to add additional value and achieve low net glucose costs. Based on the evaluation at pilot plant scale a deep insight into this new and unique technology has been generated which offers the opportunity for a short-term realization of a full scale plant for the production of low-cost fermentation sugar anywhere in the world. doi:10.1016/j.nbt.2009.06.216
2.6.114 Effect of carbon-substrates on biomass growth and recombinant protein formation in continuous cultures of Pichia pastoris Mut+ strain L. Paulova 1,∗ , M. Jan 1 , K. Melzoch 1 , J. Klein 2 , H.P. Meyer 2 , K. Kovar 3 1
Institute of Chemical Technology Prague, Prague 6, Czech Republic Lonza Ltd., Visp, Switzerland 3 Zurich University of Applied Sciences, Wadenswil, Switzerland 2
Methylotrophic yeast Pichia pastoris has been exploited in recent decades as an expression system for the production of a wide range of products. Typically, glycerol and methanol have been used in most cultivation protocols for Pichia pastoris Mut+ strains to achieve high biomass concentrations with glycerol and induce expression of a heterologous protein as well as to support biomass growth in the production phase with methanol. Limitations associated with the methanol metabolism are the considerable amount of heat released, the high demand for oxygen and, together with the need for explosion-proof facilities, simulated research on mixed-substrate strategies. Thus, glycerol, glucose, sorbitol and mixtures thereof with methanol (50% of total carbon comes from S234
www.elsevier.com/locate/nbt
methanol) were tested as alternative carbon feedstocks for the cultivation of methylotrophic yeast. Carbon-limited chemostat cultures with Pichia pastoris secreting porcine trypsinogen under the control of AOX1 promoter were performed at low dilution rates (D ∼0.03 h−1 ). After achieving a steady-state on a sole substrate, feeding followed at the same dilution rate with a mixture of the first substrate plus methanol (total carbon concentration was kept constant). The heterologous product was not expressed when cells were grown solely on glycerol or glucose. On the other hand, a low concentration of trypsinogen was detected in cultures grown solely on sorbitol even when methanol was not present due to the non-repressive character of this substrate. Grown on mixed substrates, product expression was detected shortly after methanol addition and its concentration gradually increased towards steady values of (100—200) mg l−1 depending on the type of substrate supplied with the methanol. The highest specific productivity (approx. 0.6 mg gCDW −1 h−1 ) was achieved in cultures grown on a mixture of sorbitol and methanol. doi:10.1016/j.nbt.2009.06.217
2.6.115 Phytase inactivation in the animal feed pelleting process K. Puder 1,∗ , O. Simonsen 2 , C. Jørgensen 2 , A. Jensen 3 1
Technical University of Denmark; Novozymes A/S, Kgs. Lyngby, Denmark Novozymes A/S, Denmark 3 Technical University of Denmark, Denmark 2
The addition of phytase granules into the animal diet yields in the liberation of inorganic phosphate absorb by monogastric animals. Phytase granules are complex systems composed of protein incorporated in a core of various excipients and a coating to support stability. Nevertheless, enzymatic inactivation arises during the feed processing where steam is applied and high temperatures (75—95 ◦ C) and moisture contents (up to 100%rH) are obtained. For the elucidation of the phytase deactivation pathway a small scale set up was developed providing controlled temperature, relative humidity and short-time exposure trying to mimic real pelletization conditions. Lyophilized enzyme powders and multiphase enzyme granules were studied at different process conditions and analyzed for their stability. Exposure of lyophilized enzyme powder, formulated only with Na2 SO4 treated at high temperature and omitted moisture caused already a decline of activity and increased humidity resulted in an even further drop (decrease from 60% to 5% activity). Salthydrates might have been produced or even saturated salt solution due to deliquescence impacting protein stability. A less drastic fall of activity was observed for the non-salt containing powder (from 90% to 50%). The loss of activity could be followed by a decline of the unfolding enthalpy measured by Differential Scanning Calorimetry. The inactivation seemed to be caused by an unfolding process resulting in non-covalent aggregation. The aggregation was attempted to be followed by Dynamic Light Scattering and Size Exclusion Chromatography. Various trials at different moisture levels and high temperatures were conducted with different granule types, phytase variants, and
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
prehumidified enzymes granules at pelleting relevant conditions and the activity determined. Overall, dry, hot air barely reduced the activity in the granule while exposing it to an increased relative humidity, especially after a treatment of 50%rH, the activity decreased dramatically. Phytase variants produce different levels of inactivation which allows for a stability ranking. Also prehumidifying samples, to simulate moisture uptake from the mash, followed by different treatments in the set up showed a rise in inactivation. After the melting of the coating, the moisture can get absorbed onto the granule and might diffuse into the core causing an enhanced mobility of the compounds; eventually, the alteration of the multi-phase matrix affects phytase stability. All results depicted that the combination of temperature and relative humidity is detrimental for solid-state protein stability. Eventually, the obtained stability data indicate correlation between our set up and real pelleting trials by using very high humidities (90—100%rH).
step, the protein was further purified by size exclusion chromatography (Superdex-75 10/300 GL) using buffer C (25 mM HEPES). The procedure routinely yielded 6—8 mg of pure (> 95 %) soluble bFGF per liter culture. The final product was characterized by SDS-PAGE, and identity was verified and confirmed by ESI mass spectrometry after trypsin digestion and by MALDI—TOF mass spectrometry (molecular weight expected: 22988.9 Da; molecular weight found: 22987.5 Da).
doi:10.1016/j.nbt.2009.06.218
VIT University, Jodhpur, India
2.6.116 Cloning, expression and purification of recombinant human basic fibroblast growth factor using E. Coli as expression host S. Dubey ∗ , R. Perozzo, L. Scapozza, Y. Kalia University of Geneva, Geneva, Switzerland
Basic fibroblast growth factor (bFGF; 146 amino acids) belongs to a large family of structurally related proteins that affect the growth, differentiation, migration and survival of a wide variety of cell types. Potential therapeutic applications of bFGF include tissue regeneration, wound healing and angiogenesis. The objective of this study was to develop a fast and reliable method to produce bFGF using a prokaryotic pET expression system. Here, we report on the development of a stable expression plasmid (pET100) carrying the human bfgf gene for the recombinant expression of bFGF in E. coli BL21(DE3) Star as expression host. Primers were designed to enable cleavage of the His-tag using thrombin. The primers used in the present studies were: forward primer (5 )-CAC CCT GGT GCC GCG CGG CAG CAT GGC AGC CGG GAG CAT CAC CAC GCT GCC CGC C and reverse primer (5 )-TCA GCT CTT AGC AGA CAT TGG AAG AAA AAG. The first four base pairs of the forward primer were complementary to the destination plasmid, while the bases in bold were used for coding the thrombin cleavage site. The protein was expressed in LB media at 37 ◦ C. Protein expression was induced by addition of IPTG (1 mM final concentration) to an overnight culture. Cells were harvested after 4 h of induction. Soluble protein was purified in a two stage process. In the first step, the protein was isolated and purified by Ni-affinity chromatography followed by gradient elution using buffer A (20 mM Tris, 500 mM NaCl, 10% glycerol; pH 8) and buffer B (buffer A supplemented with 500 mM imidazole) at a flow rate of 2 mL/min. The desired protein started to elute at about 30% of buffer B in buffer A. The His-tag was subsequently cleaved by incubation with thrombin (10 U enzyme per mL of protein solution) for 16 h at 16 ◦ C. As a second
doi:10.1016/j.nbt.2009.06.219
2.6.117 Growth pattern study of Spirulina platensis in seawater based media N. Bohra
Growth pattern of Spirulina platensis was investigated in standard and modified media based on seawater-chemicals and seawaterfertilizers. During the cultivation, the cell concentrations were analyzed at 540 nm along with protein and chlorophyll a estimation. Growth patterns of different species and strains were monitored for 25 days and specific growth rate, mean daily division rate and doubling time were calculated. Spirulina platensis was observed to have different specific growth characteristics in different media at same environmental parameters (Temperature, pH and light intensity). Even though, Spirulina platensis in standard media exhibited good growth patterns, biomass, protein content and chlorophyll content than other seawater based media, the experiment discusses the feasibility of seawater based media which will be a boon for developing island nations and coastal cottage industries. doi:10.1016/j.nbt.2009.06.220
2.6.118 Physiological response of yeast to process perturbations: a mini bioreactor approach A. Lejeune ∗ , F. Delvigne, H. Ghalfi, P. Thonart Gembloux Agricultural University, Gembloux, Belgium
Large-scale production of yeast (Saccharomyces cerevisiae) is difficult to control, considering the drop of mixing and mass transfer efficiency during up scaling. The drop of hydrodynamic efficiency in large-scale bioreactors induces the formation of heterogeneities, i.e. mainly substrate and dissolved oxygen in process conditions. These extracellular fluctuations exhibits have several impacts at the level of the physiology of microorganisms, from metabolic shift to specific gene expression (stress response). Microbial cell responses to extracellular fluctuations are actually not fully understood. In this work, we propose to reproduce the main extracellular fluctuations at the level of a mini bioreactor platform. These mini reactors are shake flasks equipped with dissolved oxygen probes www.elsevier.com/locate/nbt S235
ABSTRACTS
backs. Sugar cane processing sugar mills are highly integrated biorefineries producing sugar at lowest costs. Unfortunately, these sugar mills are limited to sugar cane growing regions which do not necessarily match with regions of chemical production and the corresponding chemical markets. Traditional wet mills are distributed all over the world as their raw material corn is a transportable good. As most of the produced glucose is used for human consumption, these plants are generally delivering food-quality which is not required for the fermentation industry. In addition, these facilities are highly capital-intensive which lead to large-scale facilities of 1.5 million tons corn crushing capacity to reduce the specific investment costs per ton of glucose produced. In general, this amount of glucose exceeds the demand of even a conglomerate of fermentation processes. Facing these obstacles BASF has developed a unique corn-based process for the production of low-cost fermentation-grade glucose, even at moderate scale facilities. This process has been realized at a pilot plant scale in conjunction with a fermentation and product recovery unit. Trials on the pilot plant revealed increases in titer and yield during fermentation compared to traditional sugar feedstocks. Compared to these feedstocks, the glucose solution is rich in nutrients. As a result, the amount of complex media components can be reduced, saving additional costs. The co-products generated in the process by refining of corn can be sold to the feed market to add additional value and achieve low net glucose costs. Based on the evaluation at pilot plant scale a deep insight into this new and unique technology has been generated which offers the opportunity for a short-term realization of a full scale plant for the production of low-cost fermentation sugar anywhere in the world. doi:10.1016/j.nbt.2009.06.216
2.6.114 Effect of carbon-substrates on biomass growth and recombinant protein formation in continuous cultures of Pichia pastoris Mut+ strain L. Paulova 1,∗ , M. Jan 1 , K. Melzoch 1 , J. Klein 2 , H.P. Meyer 2 , K. Kovar 3 1
Institute of Chemical Technology Prague, Prague 6, Czech Republic Lonza Ltd., Visp, Switzerland 3 Zurich University of Applied Sciences, Wadenswil, Switzerland 2
Methylotrophic yeast Pichia pastoris has been exploited in recent decades as an expression system for the production of a wide range of products. Typically, glycerol and methanol have been used in most cultivation protocols for Pichia pastoris Mut+ strains to achieve high biomass concentrations with glycerol and induce expression of a heterologous protein as well as to support biomass growth in the production phase with methanol. Limitations associated with the methanol metabolism are the considerable amount of heat released, the high demand for oxygen and, together with the need for explosion-proof facilities, simulated research on mixed-substrate strategies. Thus, glycerol, glucose, sorbitol and mixtures thereof with methanol (50% of total carbon comes from S234
www.elsevier.com/locate/nbt
methanol) were tested as alternative carbon feedstocks for the cultivation of methylotrophic yeast. Carbon-limited chemostat cultures with Pichia pastoris secreting porcine trypsinogen under the control of AOX1 promoter were performed at low dilution rates (D ∼0.03 h−1 ). After achieving a steady-state on a sole substrate, feeding followed at the same dilution rate with a mixture of the first substrate plus methanol (total carbon concentration was kept constant). The heterologous product was not expressed when cells were grown solely on glycerol or glucose. On the other hand, a low concentration of trypsinogen was detected in cultures grown solely on sorbitol even when methanol was not present due to the non-repressive character of this substrate. Grown on mixed substrates, product expression was detected shortly after methanol addition and its concentration gradually increased towards steady values of (100—200) mg l−1 depending on the type of substrate supplied with the methanol. The highest specific productivity (approx. 0.6 mg gCDW −1 h−1 ) was achieved in cultures grown on a mixture of sorbitol and methanol. doi:10.1016/j.nbt.2009.06.217
2.6.115 Phytase inactivation in the animal feed pelleting process K. Puder 1,∗ , O. Simonsen 2 , C. Jørgensen 2 , A. Jensen 3 1
Technical University of Denmark; Novozymes A/S, Kgs. Lyngby, Denmark Novozymes A/S, Denmark 3 Technical University of Denmark, Denmark 2
The addition of phytase granules into the animal diet yields in the liberation of inorganic phosphate absorb by monogastric animals. Phytase granules are complex systems composed of protein incorporated in a core of various excipients and a coating to support stability. Nevertheless, enzymatic inactivation arises during the feed processing where steam is applied and high temperatures (75—95 ◦ C) and moisture contents (up to 100%rH) are obtained. For the elucidation of the phytase deactivation pathway a small scale set up was developed providing controlled temperature, relative humidity and short-time exposure trying to mimic real pelletization conditions. Lyophilized enzyme powders and multiphase enzyme granules were studied at different process conditions and analyzed for their stability. Exposure of lyophilized enzyme powder, formulated only with Na2 SO4 treated at high temperature and omitted moisture caused already a decline of activity and increased humidity resulted in an even further drop (decrease from 60% to 5% activity). Salthydrates might have been produced or even saturated salt solution due to deliquescence impacting protein stability. A less drastic fall of activity was observed for the non-salt containing powder (from 90% to 50%). The loss of activity could be followed by a decline of the unfolding enthalpy measured by Differential Scanning Calorimetry. The inactivation seemed to be caused by an unfolding process resulting in non-covalent aggregation. The aggregation was attempted to be followed by Dynamic Light Scattering and Size Exclusion Chromatography. Various trials at different moisture levels and high temperatures were conducted with different granule types, phytase variants, and
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
prehumidified enzymes granules at pelleting relevant conditions and the activity determined. Overall, dry, hot air barely reduced the activity in the granule while exposing it to an increased relative humidity, especially after a treatment of 50%rH, the activity decreased dramatically. Phytase variants produce different levels of inactivation which allows for a stability ranking. Also prehumidifying samples, to simulate moisture uptake from the mash, followed by different treatments in the set up showed a rise in inactivation. After the melting of the coating, the moisture can get absorbed onto the granule and might diffuse into the core causing an enhanced mobility of the compounds; eventually, the alteration of the multi-phase matrix affects phytase stability. All results depicted that the combination of temperature and relative humidity is detrimental for solid-state protein stability. Eventually, the obtained stability data indicate correlation between our set up and real pelleting trials by using very high humidities (90—100%rH).
step, the protein was further purified by size exclusion chromatography (Superdex-75 10/300 GL) using buffer C (25 mM HEPES). The procedure routinely yielded 6—8 mg of pure (> 95 %) soluble bFGF per liter culture. The final product was characterized by SDS-PAGE, and identity was verified and confirmed by ESI mass spectrometry after trypsin digestion and by MALDI—TOF mass spectrometry (molecular weight expected: 22988.9 Da; molecular weight found: 22987.5 Da).
doi:10.1016/j.nbt.2009.06.218
VIT University, Jodhpur, India
2.6.116 Cloning, expression and purification of recombinant human basic fibroblast growth factor using E. Coli as expression host S. Dubey ∗ , R. Perozzo, L. Scapozza, Y. Kalia University of Geneva, Geneva, Switzerland
Basic fibroblast growth factor (bFGF; 146 amino acids) belongs to a large family of structurally related proteins that affect the growth, differentiation, migration and survival of a wide variety of cell types. Potential therapeutic applications of bFGF include tissue regeneration, wound healing and angiogenesis. The objective of this study was to develop a fast and reliable method to produce bFGF using a prokaryotic pET expression system. Here, we report on the development of a stable expression plasmid (pET100) carrying the human bfgf gene for the recombinant expression of bFGF in E. coli BL21(DE3) Star as expression host. Primers were designed to enable cleavage of the His-tag using thrombin. The primers used in the present studies were: forward primer (5 )-CAC CCT GGT GCC GCG CGG CAG CAT GGC AGC CGG GAG CAT CAC CAC GCT GCC CGC C and reverse primer (5 )-TCA GCT CTT AGC AGA CAT TGG AAG AAA AAG. The first four base pairs of the forward primer were complementary to the destination plasmid, while the bases in bold were used for coding the thrombin cleavage site. The protein was expressed in LB media at 37 ◦ C. Protein expression was induced by addition of IPTG (1 mM final concentration) to an overnight culture. Cells were harvested after 4 h of induction. Soluble protein was purified in a two stage process. In the first step, the protein was isolated and purified by Ni-affinity chromatography followed by gradient elution using buffer A (20 mM Tris, 500 mM NaCl, 10% glycerol; pH 8) and buffer B (buffer A supplemented with 500 mM imidazole) at a flow rate of 2 mL/min. The desired protein started to elute at about 30% of buffer B in buffer A. The His-tag was subsequently cleaved by incubation with thrombin (10 U enzyme per mL of protein solution) for 16 h at 16 ◦ C. As a second
doi:10.1016/j.nbt.2009.06.219
2.6.117 Growth pattern study of Spirulina platensis in seawater based media N. Bohra
Growth pattern of Spirulina platensis was investigated in standard and modified media based on seawater-chemicals and seawaterfertilizers. During the cultivation, the cell concentrations were analyzed at 540 nm along with protein and chlorophyll a estimation. Growth patterns of different species and strains were monitored for 25 days and specific growth rate, mean daily division rate and doubling time were calculated. Spirulina platensis was observed to have different specific growth characteristics in different media at same environmental parameters (Temperature, pH and light intensity). Even though, Spirulina platensis in standard media exhibited good growth patterns, biomass, protein content and chlorophyll content than other seawater based media, the experiment discusses the feasibility of seawater based media which will be a boon for developing island nations and coastal cottage industries. doi:10.1016/j.nbt.2009.06.220
2.6.118 Physiological response of yeast to process perturbations: a mini bioreactor approach A. Lejeune ∗ , F. Delvigne, H. Ghalfi, P. Thonart Gembloux Agricultural University, Gembloux, Belgium
Large-scale production of yeast (Saccharomyces cerevisiae) is difficult to control, considering the drop of mixing and mass transfer efficiency during up scaling. The drop of hydrodynamic efficiency in large-scale bioreactors induces the formation of heterogeneities, i.e. mainly substrate and dissolved oxygen in process conditions. These extracellular fluctuations exhibits have several impacts at the level of the physiology of microorganisms, from metabolic shift to specific gene expression (stress response). Microbial cell responses to extracellular fluctuations are actually not fully understood. In this work, we propose to reproduce the main extracellular fluctuations at the level of a mini bioreactor platform. These mini reactors are shake flasks equipped with dissolved oxygen probes www.elsevier.com/locate/nbt S235