- Email: [email protected]
Studies on protamine purification by surfactant precipitation
Abstracts / Journal of Biotechnology 136S (2008) S496–S505
butanediol to glucose was 676.83 when the system was composed of 25% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.7% and 90.0%, respectively. Ethanol/phosphate aqueous two-phase system is effective to the separation of 2,3-butanediol, with advantages of low cost and convenient operation. Separation of 2,3-butanediol from fermentative broth by ethanol/phosphate system was succeeded with a partition coefficient of 27.75 and recovery of 99.1%. This method provides a new possibility for the separation and refining of 2,3-butanediol. References Syu, M.J., 2001. Biological production of 2,3-butanediol. Appl. Microbiol. Biotechnol. 55, 10–18. Tan, T.W., Huo, Q., Ling, Q., 2002. Purification of glycyrrhizin from Glycyrrhiza uralensis Fisch with ethanol/phosphate aqueous two phase system. Biotechnol. Lett. 24, 1417–1420. Xiu, Z.L., Zeng, A.P., 2008. Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl. Microbiol. Biotechnol. 78, 917–926, doi:10.1007/s00253-008-13874.
doi:10.1016/j.jbiotec.2008.07.1173 V6-P-011 An integrated process of PEGylation and separation of hirudin on an anion exchange column Xueqin Li 1 , Zhilong Xiu 1,∗ , Jun Zhao 1 , Shirong Li 1 , Xiaohui Li 1 , Zhiguo Su 2 1 Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian, PR China 2 Institute of Process Engineering, Chinese Academy of Science, Beijing, PR China
E-mail address: [email protected] (Z. Xiu). Hirudin, with most potent antithrombin activity, is limited in use for its short circulation half-life. PEGylation is an effective way to increase the stabilities and prolong the half-life of hirudin To obtain highly homogenate mono-PEG-hirudin with high-anticoagulant activity, an anion exchange column was applied to assist the reaction between hirudin and PEG. In this study a recombinant hirudin variant 2 (HV2) was adopted to be PEGylated by using SC-mPEG 5 kDa and SC-mPEG 20 kDa on an anion exchange column. It proved that on-column PEGylation could promote the homogeneity of the PEGylated products and enhance remarkably the in vitro anticoagulant activity of mono-PEG-hirudin compared with PEGylation in solution phase. For instance, the in vitro-specific anticoagulant activities retained to native hirudin increased from 26%, 55% PEGylated in solution phase to 91%, 96% on column for mono-PEG 5 kDa-hirudin and mono-PEG 20 kDa-hirudin, respectively. A large size of PEG seems to be favorable for stability of hirudin activity and site-specific PEGylation of hirudin. The optimal PEGylation of HV2 with SC-mPEG 20 kDa on column was achieved for 1 h at pH 8.0 in a molar ratio of mPEG/HV2 of 9:1. The molecular dynamics simulation and analysis showed that the 35th lysine residue of hirudin was likely to be modified by SC-mPEG on column. References Hou, B.B., Li, X.H., Xiu, Z.L., 2007. Design, reparation and in vitro bioactivity of monoPEGylated recombinant hirudin. Chin. J. Chem. Eng. 15 (6), 775–780. Lee, E.K., Lee, J.D., 2004. Proceedings of the International Symposium on the Separation of Proteins, Peptides and Polynucleotides. Federal Republic of Germany, Aachen, pp. 19–22.
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Qin, H.N., Xiu, Z.L., Zhang, D.J., Bao, Y.M., Li, X.H., Han, G.Z., 2007. PEGylation of hirudin and analysis of its antithrombin activity in vitro. Chin. J. Chem. Eng. 15 (4), 586–590.
doi:10.1016/j.jbiotec.2008.07.1174 V6-P-012 Removal of cells from 2,3-butanediol fermentation broth by flocculation and reuse of cells in flocs Jianghong Zhang, Lihui Sun, Zhilong Xiu ∗ Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116023, China E-mail address: [email protected] (Z. Xiu). 2,3-Butanediol is a colorless and odorless product with extensive industrial application. It is a potential valuable fuel additive for its high boiling point. It can also be used as a drug carrier. Besides, it is used in cosmetic products, lotions and antifreeze agents. A preferred way to produce 2,3-butanediol is bioconversion. However, a key factor affecting its cost is the downstream process of fermentation broth. Recently, the flocculation of fermentation broth has been studied widely, but its application on 2,3-butanediol has not been reported yet. In this paper, the pretreatment of 2,3-butanediol fermentation broth by chitosan flocculation was investigated. Using sodium alginate as coagulant, the effects of chitosan molecular weight, chitosan dosage, coagulant aid dosage, pH and turbidity time on the flocculation of fermentation broth were investigated, respectively. The performance was evaluated by cell removal ratio. According to the results, the optional flocculation conditions for 2,3-butanediol are as follows: 0.375 g/L of chitoson with 40 kDa molecular weight, 0.250 g/L of sodium alginate, 5.0 of pH, 30 min of turbidity time and 1 h of settlement time. The removal ratio of cells can reach 98%, the retained ratio of 2,3-butanediol can reach 99%, and the flocculated broth is clear. After flocculation the cells of Klebsiella pneumoniae in flocs can grow well (OD 13.5) and be used repeatedly. References Cheng, K.K., Liu, H.J., Liu, D.H., 2005. Multiple growth inhibition of Klebsiella pneumoniae in 1,3-propanediol fermentation. Biotechnol. Lett. 27, 19–22. Helander, I.M., Nurmiaho-Lassila, E.L., Ahvenainen, R., 2001. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. Int. J. Microbiol. 71, 235–244. Qin, J.Y., Xiao, Z.J., Ma, C.Q., 2006. Production of 2,3-butanediol by K. pneumoniae using glucose and ammonium phosphate. Chinese J. Chem. Eng. 14 (1), 132–136. Sun, Z.J., Lv, G.J., Li, S.Y., 2007. Probing the role of microenvironment for microencapsulated Sacchromyces cerevisiae under osmotic stress. J. Biotechnol. 128, 150–161. Syu, M.J., 2001. Biological production of 2,3-butanediol. Appl. Microbiol. Biotechnol. 55, 10–18.
doi:10.1016/j.jbiotec.2008.07.1175 V6-P-013 Studies on protamine purification by surfactant precipitation Junguo Liu ∗ , Du Liye, Zhao Zihua, Wei Nana College of Bioscience and Bioengineering, Hebei University of Science and Technology, Hebei Province, 050018, PR China E-mail address: [email protected] (J. Liu). Protamine is a cationic peptide originated from fish milt and used as a carrier for injectable insulin, a heparin antagonist in medical field and as an antibacterial ingredient in some food products.
S502
Abstracts / Journal of Biotechnology 136S (2008) S496–S505
Although protamine is produced commercially, the purification information is unavailable and the published methods in the literature usually involve expensive chromatographic steps and are time consuming. In this paper, for the first time, surfactant precipitation was used to capture protamine from aqueous solution. The precipitation and recovery process were investigated in order to set up a novel purification method for protamine production. Firstly, two surfactants, sodium dodecyl sulfate (SDS) and sodium di(2-ethylhexyl) sulfosuccinate (AOT) were investigated to precipitate protamine. The results showed that protamine formed a water insoluble complex upon contact with whether AOT or SDS, and precipitated from the aqueous solution. The electrostatic interaction between cationic protamine and anionic surfactant could be the reason for the formation of protamine–surfactant complex. Surfactants contribute the high hydrophobicity of protamine–surfactant complex. The two points are suggested to explain the precipitation of protmaine–surfactant complex. The molar ratio in the protamine–SDS complex is changing with the quantity of SDS added. So it is with protamine–AOT complex. What is more interesting, 100% protamine precipitation is achieved by increasing SDS added to the protamine solution. But the precipitation curve against SDS has a shape of ‘Bell’, which means that the optimal precipitation rate is about 74.6%, and the precipitation rate went down when more or less SDS was added. Further organic solvent and a small amount of NaCl solution were added to the complex of protamine–surfactant to dissolve the surfactant into the organic solvent phase and recover protamine. It was demonstrated that the 1-propanol is excellent for protamine–SDS complex, while acetone is optimal for protamine–AOT complex. The recovered protamine was free of surfactant and retained its original protein activity. The solvent volume, NaCl concentration were optimized.
References Ando, T., Yamasaki, M., Suzuki, K., 1973. Protamines: Isolation, Characterization, Structure and Function. Springer-Verlag, Berlin. Tom A. Gill, Douglas S. Singer, John W. Thompson, 2006. Purification and analysis of protamine. Process Biochem. 41, 1875–1882. Sakaguchi, S., 1950. A new method for the colorimetric determination of arginine. J. Biochem. 37, 231–236. Shin, Y.-O., Weber, M.E., Vera, J.H., 2003. Reverse micellar extraction and precipitation of lysozyme using sodium di(2-ethylhexyl) sulfosuccinate. Biotechnol. Prog. 19, 928–935. Shin, Y.-O., Rodil, E., Vera, J.H., 2004. Surfactant precipitation and polar solvent recovery of alpha-chymotrypsin and ribonuclease-A. Biochem. Eng. J. 17, 91–97.
aimed at identifying the feasibility and generic applicability of directly integrating cell disruption by HPH and product capture by aqueous two-phase extraction (ATPE) for in situ extraction of intracellular glucose oxidase. One important requirement of ATPE is that the contaminant proteins should not be extracted in the target enzyme-rich phase. Hence, along with a high Kg (for glucose oxidase), a low Kp (for contaminated protein) is desirable. EO40 PO60 2800 gave fairly high Kg of 23.2 and the least Kp (0.8) and gave the highest purification factor (3.49). Although the current ATPE could extract 88.5% of glucose oxidase contained in the clarified disruptate, only 46.1% of the total enzyme could be recovered after ATPE. In the in situ ATPE, an improved yield of 67.8% could be obtained and a higher specific acitivity of 90.2 U/mg protein was achieved. In the current ATPE, the activity of glucose oxidase was lost during cell disruption and following disruptate clarification, mainly due to the interaction of debris–protein as well as the cellular and/or molecular degradation. However, clarification and temporary storage of the disruptate was eliminated in the in situ ATPE process. Glucose oxidase could move into the EO40 PO60 -rich phase, once releasing by cell disruption. Thus the exposure of glucose oxidase to potential antagonists (debris adsorbents, degrading enzymes) was minimized due to the different partitioning behaviours of glucose oxidase and potential antagonists. Moreover, the high extraction ability of ATPE realized the immediate separation of glucose oxidase from cell debris, resulting in the reduced amount of glucose oxidase adsorbed by cell debris.
References Desai, M.A., 2000. Downstream Processing of Proteins: Methods and Protocols. Humana Press, Totowa. Su, Z.G., Feng, X.L., 1999. Process integration of cell disruption and aqueous twophase extraction. J. Chem. Technol. Biotechnol. 74, 284–288. Zhu, J.H., Yan, X.L., Chen, H.J., Wang, Z.H., 2007. In situ extraction of intracellular l-asparaginase using thermoseparating aqueous two-phase systems. J. Chromatogr. A 1147, 127–1334.
doi:10.1016/j.jbiotec.2008.07.1177 V6-P-018 Application of mechanochemical pretreatment (MCPT) to aqueous extraction of eleutheroside b from Eleutherococcus senticosus
doi:10.1016/j.jbiotec.2008.07.1176
Chunna Song ∗ , Liji Jin, Yongping Xu
V6-P-017
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, China
In situ extraction of intracellular protein combined with highpressure homogenization (HPH)
E-mail address: [email protected] (C. Song).
Jian-Hang Zhu 1,∗ , Bao Zhang 2 1
Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China 2 Department of Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China E-mail address: [email protected] (J.-H. Zhu).
Technologists are in search of and the industry is in demand of efficient yet inexpensive large-scale separation techniques with short process times for intracellular proteins. In situ extraction, also called an integrated release–extraction process in downstream techniques, provides an innocuous environment for separation and purification of protein, in which target product release takes place and immediate product extraction follows. The present study
A mechaochemical pretreatment (MCPT) was applied to aqueous extraction of eleutheroside B from the Asian herb, Eleutherococcus senticosus. To optimize the mechanochemical assisted extraction (MCAE) on eleutheroside B, the MCAE process was optimized based on both orthogonal and one-factor experiment and was compared with conventional heat reflux extraction. Both particle analysis and scanning electron micrographs suggested that the plant cell wall could be broken up after the mechanochemcal treatment. The optimum parameters were as follows: HP--CD content, 23% (w/w); milling period, 25 min; extraction solvent, water; circulating water temperature, 40 ◦ C. The results of both MS analysis and recovery experiments showed that MCAE is reliable and accurate for eleutheroside B extraction. Under these parameters, the eleutheroside B yield increased by 32.5% comparing with heatreflux extraction. In the specific case of eleutheroside B, significant
butanediol to glucose was 676.83 when the system was composed of 25% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.7% and 90.0%, respectively. Ethanol/phosphate aqueous two-phase system is effective to the separation of 2,3-butanediol, with advantages of low cost and convenient operation. Separation of 2,3-butanediol from fermentative broth by ethanol/phosphate system was succeeded with a partition coefficient of 27.75 and recovery of 99.1%. This method provides a new possibility for the separation and refining of 2,3-butanediol. References Syu, M.J., 2001. Biological production of 2,3-butanediol. Appl. Microbiol. Biotechnol. 55, 10–18. Tan, T.W., Huo, Q., Ling, Q., 2002. Purification of glycyrrhizin from Glycyrrhiza uralensis Fisch with ethanol/phosphate aqueous two phase system. Biotechnol. Lett. 24, 1417–1420. Xiu, Z.L., Zeng, A.P., 2008. Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl. Microbiol. Biotechnol. 78, 917–926, doi:10.1007/s00253-008-13874.
doi:10.1016/j.jbiotec.2008.07.1173 V6-P-011 An integrated process of PEGylation and separation of hirudin on an anion exchange column Xueqin Li 1 , Zhilong Xiu 1,∗ , Jun Zhao 1 , Shirong Li 1 , Xiaohui Li 1 , Zhiguo Su 2 1 Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian, PR China 2 Institute of Process Engineering, Chinese Academy of Science, Beijing, PR China
E-mail address: [email protected] (Z. Xiu). Hirudin, with most potent antithrombin activity, is limited in use for its short circulation half-life. PEGylation is an effective way to increase the stabilities and prolong the half-life of hirudin To obtain highly homogenate mono-PEG-hirudin with high-anticoagulant activity, an anion exchange column was applied to assist the reaction between hirudin and PEG. In this study a recombinant hirudin variant 2 (HV2) was adopted to be PEGylated by using SC-mPEG 5 kDa and SC-mPEG 20 kDa on an anion exchange column. It proved that on-column PEGylation could promote the homogeneity of the PEGylated products and enhance remarkably the in vitro anticoagulant activity of mono-PEG-hirudin compared with PEGylation in solution phase. For instance, the in vitro-specific anticoagulant activities retained to native hirudin increased from 26%, 55% PEGylated in solution phase to 91%, 96% on column for mono-PEG 5 kDa-hirudin and mono-PEG 20 kDa-hirudin, respectively. A large size of PEG seems to be favorable for stability of hirudin activity and site-specific PEGylation of hirudin. The optimal PEGylation of HV2 with SC-mPEG 20 kDa on column was achieved for 1 h at pH 8.0 in a molar ratio of mPEG/HV2 of 9:1. The molecular dynamics simulation and analysis showed that the 35th lysine residue of hirudin was likely to be modified by SC-mPEG on column. References Hou, B.B., Li, X.H., Xiu, Z.L., 2007. Design, reparation and in vitro bioactivity of monoPEGylated recombinant hirudin. Chin. J. Chem. Eng. 15 (6), 775–780. Lee, E.K., Lee, J.D., 2004. Proceedings of the International Symposium on the Separation of Proteins, Peptides and Polynucleotides. Federal Republic of Germany, Aachen, pp. 19–22.
S501
Qin, H.N., Xiu, Z.L., Zhang, D.J., Bao, Y.M., Li, X.H., Han, G.Z., 2007. PEGylation of hirudin and analysis of its antithrombin activity in vitro. Chin. J. Chem. Eng. 15 (4), 586–590.
doi:10.1016/j.jbiotec.2008.07.1174 V6-P-012 Removal of cells from 2,3-butanediol fermentation broth by flocculation and reuse of cells in flocs Jianghong Zhang, Lihui Sun, Zhilong Xiu ∗ Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116023, China E-mail address: [email protected] (Z. Xiu). 2,3-Butanediol is a colorless and odorless product with extensive industrial application. It is a potential valuable fuel additive for its high boiling point. It can also be used as a drug carrier. Besides, it is used in cosmetic products, lotions and antifreeze agents. A preferred way to produce 2,3-butanediol is bioconversion. However, a key factor affecting its cost is the downstream process of fermentation broth. Recently, the flocculation of fermentation broth has been studied widely, but its application on 2,3-butanediol has not been reported yet. In this paper, the pretreatment of 2,3-butanediol fermentation broth by chitosan flocculation was investigated. Using sodium alginate as coagulant, the effects of chitosan molecular weight, chitosan dosage, coagulant aid dosage, pH and turbidity time on the flocculation of fermentation broth were investigated, respectively. The performance was evaluated by cell removal ratio. According to the results, the optional flocculation conditions for 2,3-butanediol are as follows: 0.375 g/L of chitoson with 40 kDa molecular weight, 0.250 g/L of sodium alginate, 5.0 of pH, 30 min of turbidity time and 1 h of settlement time. The removal ratio of cells can reach 98%, the retained ratio of 2,3-butanediol can reach 99%, and the flocculated broth is clear. After flocculation the cells of Klebsiella pneumoniae in flocs can grow well (OD 13.5) and be used repeatedly. References Cheng, K.K., Liu, H.J., Liu, D.H., 2005. Multiple growth inhibition of Klebsiella pneumoniae in 1,3-propanediol fermentation. Biotechnol. Lett. 27, 19–22. Helander, I.M., Nurmiaho-Lassila, E.L., Ahvenainen, R., 2001. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. Int. J. Microbiol. 71, 235–244. Qin, J.Y., Xiao, Z.J., Ma, C.Q., 2006. Production of 2,3-butanediol by K. pneumoniae using glucose and ammonium phosphate. Chinese J. Chem. Eng. 14 (1), 132–136. Sun, Z.J., Lv, G.J., Li, S.Y., 2007. Probing the role of microenvironment for microencapsulated Sacchromyces cerevisiae under osmotic stress. J. Biotechnol. 128, 150–161. Syu, M.J., 2001. Biological production of 2,3-butanediol. Appl. Microbiol. Biotechnol. 55, 10–18.
doi:10.1016/j.jbiotec.2008.07.1175 V6-P-013 Studies on protamine purification by surfactant precipitation Junguo Liu ∗ , Du Liye, Zhao Zihua, Wei Nana College of Bioscience and Bioengineering, Hebei University of Science and Technology, Hebei Province, 050018, PR China E-mail address: [email protected] (J. Liu). Protamine is a cationic peptide originated from fish milt and used as a carrier for injectable insulin, a heparin antagonist in medical field and as an antibacterial ingredient in some food products.
S502
Abstracts / Journal of Biotechnology 136S (2008) S496–S505
Although protamine is produced commercially, the purification information is unavailable and the published methods in the literature usually involve expensive chromatographic steps and are time consuming. In this paper, for the first time, surfactant precipitation was used to capture protamine from aqueous solution. The precipitation and recovery process were investigated in order to set up a novel purification method for protamine production. Firstly, two surfactants, sodium dodecyl sulfate (SDS) and sodium di(2-ethylhexyl) sulfosuccinate (AOT) were investigated to precipitate protamine. The results showed that protamine formed a water insoluble complex upon contact with whether AOT or SDS, and precipitated from the aqueous solution. The electrostatic interaction between cationic protamine and anionic surfactant could be the reason for the formation of protamine–surfactant complex. Surfactants contribute the high hydrophobicity of protamine–surfactant complex. The two points are suggested to explain the precipitation of protmaine–surfactant complex. The molar ratio in the protamine–SDS complex is changing with the quantity of SDS added. So it is with protamine–AOT complex. What is more interesting, 100% protamine precipitation is achieved by increasing SDS added to the protamine solution. But the precipitation curve against SDS has a shape of ‘Bell’, which means that the optimal precipitation rate is about 74.6%, and the precipitation rate went down when more or less SDS was added. Further organic solvent and a small amount of NaCl solution were added to the complex of protamine–surfactant to dissolve the surfactant into the organic solvent phase and recover protamine. It was demonstrated that the 1-propanol is excellent for protamine–SDS complex, while acetone is optimal for protamine–AOT complex. The recovered protamine was free of surfactant and retained its original protein activity. The solvent volume, NaCl concentration were optimized.
References Ando, T., Yamasaki, M., Suzuki, K., 1973. Protamines: Isolation, Characterization, Structure and Function. Springer-Verlag, Berlin. Tom A. Gill, Douglas S. Singer, John W. Thompson, 2006. Purification and analysis of protamine. Process Biochem. 41, 1875–1882. Sakaguchi, S., 1950. A new method for the colorimetric determination of arginine. J. Biochem. 37, 231–236. Shin, Y.-O., Weber, M.E., Vera, J.H., 2003. Reverse micellar extraction and precipitation of lysozyme using sodium di(2-ethylhexyl) sulfosuccinate. Biotechnol. Prog. 19, 928–935. Shin, Y.-O., Rodil, E., Vera, J.H., 2004. Surfactant precipitation and polar solvent recovery of alpha-chymotrypsin and ribonuclease-A. Biochem. Eng. J. 17, 91–97.
aimed at identifying the feasibility and generic applicability of directly integrating cell disruption by HPH and product capture by aqueous two-phase extraction (ATPE) for in situ extraction of intracellular glucose oxidase. One important requirement of ATPE is that the contaminant proteins should not be extracted in the target enzyme-rich phase. Hence, along with a high Kg (for glucose oxidase), a low Kp (for contaminated protein) is desirable. EO40 PO60 2800 gave fairly high Kg of 23.2 and the least Kp (0.8) and gave the highest purification factor (3.49). Although the current ATPE could extract 88.5% of glucose oxidase contained in the clarified disruptate, only 46.1% of the total enzyme could be recovered after ATPE. In the in situ ATPE, an improved yield of 67.8% could be obtained and a higher specific acitivity of 90.2 U/mg protein was achieved. In the current ATPE, the activity of glucose oxidase was lost during cell disruption and following disruptate clarification, mainly due to the interaction of debris–protein as well as the cellular and/or molecular degradation. However, clarification and temporary storage of the disruptate was eliminated in the in situ ATPE process. Glucose oxidase could move into the EO40 PO60 -rich phase, once releasing by cell disruption. Thus the exposure of glucose oxidase to potential antagonists (debris adsorbents, degrading enzymes) was minimized due to the different partitioning behaviours of glucose oxidase and potential antagonists. Moreover, the high extraction ability of ATPE realized the immediate separation of glucose oxidase from cell debris, resulting in the reduced amount of glucose oxidase adsorbed by cell debris.
References Desai, M.A., 2000. Downstream Processing of Proteins: Methods and Protocols. Humana Press, Totowa. Su, Z.G., Feng, X.L., 1999. Process integration of cell disruption and aqueous twophase extraction. J. Chem. Technol. Biotechnol. 74, 284–288. Zhu, J.H., Yan, X.L., Chen, H.J., Wang, Z.H., 2007. In situ extraction of intracellular l-asparaginase using thermoseparating aqueous two-phase systems. J. Chromatogr. A 1147, 127–1334.
doi:10.1016/j.jbiotec.2008.07.1177 V6-P-018 Application of mechanochemical pretreatment (MCPT) to aqueous extraction of eleutheroside b from Eleutherococcus senticosus
doi:10.1016/j.jbiotec.2008.07.1176
Chunna Song ∗ , Liji Jin, Yongping Xu
V6-P-017
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, China
In situ extraction of intracellular protein combined with highpressure homogenization (HPH)
E-mail address: [email protected] (C. Song).
Jian-Hang Zhu 1,∗ , Bao Zhang 2 1
Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China 2 Department of Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China E-mail address: [email protected] (J.-H. Zhu).
Technologists are in search of and the industry is in demand of efficient yet inexpensive large-scale separation techniques with short process times for intracellular proteins. In situ extraction, also called an integrated release–extraction process in downstream techniques, provides an innocuous environment for separation and purification of protein, in which target product release takes place and immediate product extraction follows. The present study
A mechaochemical pretreatment (MCPT) was applied to aqueous extraction of eleutheroside B from the Asian herb, Eleutherococcus senticosus. To optimize the mechanochemical assisted extraction (MCAE) on eleutheroside B, the MCAE process was optimized based on both orthogonal and one-factor experiment and was compared with conventional heat reflux extraction. Both particle analysis and scanning electron micrographs suggested that the plant cell wall could be broken up after the mechanochemcal treatment. The optimum parameters were as follows: HP--CD content, 23% (w/w); milling period, 25 min; extraction solvent, water; circulating water temperature, 40 ◦ C. The results of both MS analysis and recovery experiments showed that MCAE is reliable and accurate for eleutheroside B extraction. Under these parameters, the eleutheroside B yield increased by 32.5% comparing with heatreflux extraction. In the specific case of eleutheroside B, significant