- Email: [email protected]
An integrated process of PEGylation and separation of hirudin on an anion exchange column
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.
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.
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.