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Efficient production of low molecular weight heparin (LMWH) by fusion protein of MBP-heparinase I
Abstracts / Journal of Biotechnology 136S (2008) S356–S401
Four oxynitrilases from Almond, Peach, Black plum and Apple were used in the synthesis of (R)-(−)-1-(2-naphthyl)-2-(N-methyl) aminoethanol (Liu et al., 2005), an analogue of epinephrine. The yield (68%) and ee (88.3%) of the (R)-cyanohydrin generated from 2-naphaldehyde catalyzed by Almond meal were higher than the three others. The O-protected derivative acetate of the cyanohydrin was transformed to (R)-(−)-1-(2-naphthyl)-2-(Nmethyl) aminoethanol with 47% total yield and 88.0% ee through reduction by LiAlH4 , formylation by ethyl formate and reduction by LiAlH4 . In conclusion, a method for the chemo-enzymatic synthesis of chiral aminoalcohol drug compounds was developed. References Han, S.Q., Ouyang, P.K., Wei, P., Hu, Y., 2006. Enzymatic synthesis of (R)-cyanohydrins by novel (R)-oxynitrilase from Vicia sativa L. Biotechnol. Lett. 28 (23), 1909–1912. Liu, W.M., Song, R.J., Zhang, S.Y., 2005. Synthesis of (R)-(−)-1-(2-naphthyl)-2-(Nmethyl) aminoethanol. Chin. J. Med. Chem. 15 (5), 271–273. Marek, Z., Agnieszka, T.K., Andrzej, P.K., Aldona, C., 2003. Asymmetric synthesis of (S)-bufuralol and a propafenone analogue. Tetrahedron: Asymmetry 14 (12), 1659–1664. Marek, Z., Agnieszka, T.K., Andrzej, P.K., 2005. Enantioselective reduction of benzofuryl halomethyl ketones: asymmetric synthesis of (R)-bufuralol. Tetrahedron: Asymmetry 16 (19), 3205–3210.
doi:10.1016/j.jbiotec.2008.07.917 V3-YP-001 Efficient production of low molecular weight heparin (LMWH) by fusion protein of MBP-heparinase I Fengchun Ye ∗ , Shuo Chen, Yuan Xue, Yin Chen, Ying Kuang, Xinhui Kuang Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China E-mail address: [email protected] (F. Ye). For the side effects of heparin as the anticoagulant and antithrombotic agent, low molecular weight heparin (LMWH) capable of maintaining the anticoagulant activity while showing less side effect has been widely used (Kuang et al., 2006). LMWHs can be produced by controlled chemical or enzymatic breakdown of the heparin polymer. Up to now, enzymatic depolymerization of heparin has been achieved by heparinase I (EC 4.2.2.7). In order to solve the purification problem, an efficient expression system to produce soluble and active heparinase I in recombinant Escherichia coli for the enzymatic preparation of LMWH by fusion to maltosebinding protein (MBP) was constructed (Chen et al., 2005). The use of MBP-HepA showed that it degraded heparin effectively and the reaction time was a key factor in controlling the average molecular weight of the products. A membrane enzyme reactor consisting of a UF membrane was designed, in which crude enzyme was added and the enzymatic reaction was stopped by adding acid when the average absorbance at 235 nm reached different values (20–80), and then the reacted mixtures were all filtrated to obtain different LMWHs. Different indexes of the LMWHs were tested including the weight-average molecular weight (Mw ), the number-average molecular weight (Mn ) (detected by the HPLC method), the yield, the anti-factor Xa activity and the antifactor IIa activity. The results suggested that the LMWH product obtained from the mixture when the average absorbance at 235 nm reached 50 would give a satisfied result according to the European Pharmacopoeia 5.0.
S397
References Chen, Y., Xing, X.H., Lou, K., 2005. Construction of recombinant Escherichia coli for over-production of soluble heparinase I by fusion to maltose-binding protein. Biochem. Eng. J. 23, 155–159. Kuang, Y., Xing, X.H., Chen, Y., Ye, F.C., et al., 2006. Production of heparin oligosaccharides by fusion protein of MBP-heparinase I and the enzyme thermostability. J. Mol. Catal. B-Enzym. 43, 90–95.
doi:10.1016/j.jbiotec.2008.07.918 V3-YP-034 Study on the kinetic characteristics of asymmetric reduction of 2,5-hexanedione to (2S,5S)-2,5-hexanediol with yeast cells Meitian Xiao 1,2,∗ , Yawu Zhang 2 , Shaohong Lian 2 , Jing Ye 2 , Yayan Huang 2 1
Institute of Pharmaceutical Engineering, Huaqiao University, Quanzhou 362021, China 2 Department of Chemical and Pharmaceutical Engineering, Huaqiao University, Quanzhou 362021, China E-mail address: [email protected] (M. Xiao). (2S,5S)-2,5-Hexanediol is an important building block for chiral phosphine catalysts and fine chemicals, particularly pharmaceuticals and agrochemicals (Kim et al., 2005; Cobley et al., 2001). Currently, (2S,5S)- and (2R,5R)-hexanediol are synthesized on an industrial scale by lipase-catalyzed transesterification starting from the racemic/meso mixture (2,5)-hexanediol. Asymmetric reduction of carbonyl compounds by baker’s yeast (Saccharmyces cerevisiae) has been well documented and often provides a convenient route to enantiomerically pure secondary alcohols (Nakamura et al., 2003). Here, asymmetric reduction of diketones by baker’s yeast was investigated, (2S,5S)-hexanediol was produced staring from 2,5-hexanedione by baker’s yeast. Quantification of 2,5-hexanedione, 5-hydroxyhexane-2-one and 2,5-hexanediol in the fermentation was carried out using gas chromatograph with a INNOWAX column, and 1,3-propanediol as an internal standard. Determination of enantiomeric excess was performed on a HP-6890N GC with an AE. SE-30 column after the analytes were derivatized. The sp. strain S.c. No. 6 was screened from 20 strains of Saccharmyces cerevisiae, which showed higher reduction activity and enantioselectivity for the bioreduction of the substrate, and the product was mainly (2S,5S)-2,5-hexandiol. The 5hydroxyhexane-2-one in the fermentation was identified by GC-MS and it was presumed that (S)-5-hydroxyhexane-2-one was followed by (2S,5S)-2,5-hexanediol in the process of stereoselective reduction of 2,5-hexanedione. The influence of the biochemical factors on the asymmetric reduction of 2,5-hexandione catalyzed by yeast S.c. No. 6 was investigated. It was then confirmed that the appropriate condition was 34 ◦ C, pH 7.0, cell concentration 50 g L−1 (cell dry weight), initial glucose concentration 20 g L−1 , the initial substrate concentration 30 mmol L−1 , cultivation 28 h, the yield was 78.7%, and the enantiomeric excess was 94.4% for (2S,5S)2,5-hexandiol. The experimental results, including the isolation of the special strain with high reduction activity and the optimization of bioreduction conditions presented the foundation for the further scale-up experiment. References Cobley, C.J., Lennon, I.C., Mc, C.R., 2001. On the economic application of DuPHOS rhodium(I) catalysts: a comparison of COD versus NBD precatalysts. Tetrahedron Lett. 42, 7481–7483.
Four oxynitrilases from Almond, Peach, Black plum and Apple were used in the synthesis of (R)-(−)-1-(2-naphthyl)-2-(N-methyl) aminoethanol (Liu et al., 2005), an analogue of epinephrine. The yield (68%) and ee (88.3%) of the (R)-cyanohydrin generated from 2-naphaldehyde catalyzed by Almond meal were higher than the three others. The O-protected derivative acetate of the cyanohydrin was transformed to (R)-(−)-1-(2-naphthyl)-2-(Nmethyl) aminoethanol with 47% total yield and 88.0% ee through reduction by LiAlH4 , formylation by ethyl formate and reduction by LiAlH4 . In conclusion, a method for the chemo-enzymatic synthesis of chiral aminoalcohol drug compounds was developed. References Han, S.Q., Ouyang, P.K., Wei, P., Hu, Y., 2006. Enzymatic synthesis of (R)-cyanohydrins by novel (R)-oxynitrilase from Vicia sativa L. Biotechnol. Lett. 28 (23), 1909–1912. Liu, W.M., Song, R.J., Zhang, S.Y., 2005. Synthesis of (R)-(−)-1-(2-naphthyl)-2-(Nmethyl) aminoethanol. Chin. J. Med. Chem. 15 (5), 271–273. Marek, Z., Agnieszka, T.K., Andrzej, P.K., Aldona, C., 2003. Asymmetric synthesis of (S)-bufuralol and a propafenone analogue. Tetrahedron: Asymmetry 14 (12), 1659–1664. Marek, Z., Agnieszka, T.K., Andrzej, P.K., 2005. Enantioselective reduction of benzofuryl halomethyl ketones: asymmetric synthesis of (R)-bufuralol. Tetrahedron: Asymmetry 16 (19), 3205–3210.
doi:10.1016/j.jbiotec.2008.07.917 V3-YP-001 Efficient production of low molecular weight heparin (LMWH) by fusion protein of MBP-heparinase I Fengchun Ye ∗ , Shuo Chen, Yuan Xue, Yin Chen, Ying Kuang, Xinhui Kuang Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China E-mail address: [email protected] (F. Ye). For the side effects of heparin as the anticoagulant and antithrombotic agent, low molecular weight heparin (LMWH) capable of maintaining the anticoagulant activity while showing less side effect has been widely used (Kuang et al., 2006). LMWHs can be produced by controlled chemical or enzymatic breakdown of the heparin polymer. Up to now, enzymatic depolymerization of heparin has been achieved by heparinase I (EC 4.2.2.7). In order to solve the purification problem, an efficient expression system to produce soluble and active heparinase I in recombinant Escherichia coli for the enzymatic preparation of LMWH by fusion to maltosebinding protein (MBP) was constructed (Chen et al., 2005). The use of MBP-HepA showed that it degraded heparin effectively and the reaction time was a key factor in controlling the average molecular weight of the products. A membrane enzyme reactor consisting of a UF membrane was designed, in which crude enzyme was added and the enzymatic reaction was stopped by adding acid when the average absorbance at 235 nm reached different values (20–80), and then the reacted mixtures were all filtrated to obtain different LMWHs. Different indexes of the LMWHs were tested including the weight-average molecular weight (Mw ), the number-average molecular weight (Mn ) (detected by the HPLC method), the yield, the anti-factor Xa activity and the antifactor IIa activity. The results suggested that the LMWH product obtained from the mixture when the average absorbance at 235 nm reached 50 would give a satisfied result according to the European Pharmacopoeia 5.0.
S397
References Chen, Y., Xing, X.H., Lou, K., 2005. Construction of recombinant Escherichia coli for over-production of soluble heparinase I by fusion to maltose-binding protein. Biochem. Eng. J. 23, 155–159. Kuang, Y., Xing, X.H., Chen, Y., Ye, F.C., et al., 2006. Production of heparin oligosaccharides by fusion protein of MBP-heparinase I and the enzyme thermostability. J. Mol. Catal. B-Enzym. 43, 90–95.
doi:10.1016/j.jbiotec.2008.07.918 V3-YP-034 Study on the kinetic characteristics of asymmetric reduction of 2,5-hexanedione to (2S,5S)-2,5-hexanediol with yeast cells Meitian Xiao 1,2,∗ , Yawu Zhang 2 , Shaohong Lian 2 , Jing Ye 2 , Yayan Huang 2 1
Institute of Pharmaceutical Engineering, Huaqiao University, Quanzhou 362021, China 2 Department of Chemical and Pharmaceutical Engineering, Huaqiao University, Quanzhou 362021, China E-mail address: [email protected] (M. Xiao). (2S,5S)-2,5-Hexanediol is an important building block for chiral phosphine catalysts and fine chemicals, particularly pharmaceuticals and agrochemicals (Kim et al., 2005; Cobley et al., 2001). Currently, (2S,5S)- and (2R,5R)-hexanediol are synthesized on an industrial scale by lipase-catalyzed transesterification starting from the racemic/meso mixture (2,5)-hexanediol. Asymmetric reduction of carbonyl compounds by baker’s yeast (Saccharmyces cerevisiae) has been well documented and often provides a convenient route to enantiomerically pure secondary alcohols (Nakamura et al., 2003). Here, asymmetric reduction of diketones by baker’s yeast was investigated, (2S,5S)-hexanediol was produced staring from 2,5-hexanedione by baker’s yeast. Quantification of 2,5-hexanedione, 5-hydroxyhexane-2-one and 2,5-hexanediol in the fermentation was carried out using gas chromatograph with a INNOWAX column, and 1,3-propanediol as an internal standard. Determination of enantiomeric excess was performed on a HP-6890N GC with an AE. SE-30 column after the analytes were derivatized. The sp. strain S.c. No. 6 was screened from 20 strains of Saccharmyces cerevisiae, which showed higher reduction activity and enantioselectivity for the bioreduction of the substrate, and the product was mainly (2S,5S)-2,5-hexandiol. The 5hydroxyhexane-2-one in the fermentation was identified by GC-MS and it was presumed that (S)-5-hydroxyhexane-2-one was followed by (2S,5S)-2,5-hexanediol in the process of stereoselective reduction of 2,5-hexanedione. The influence of the biochemical factors on the asymmetric reduction of 2,5-hexandione catalyzed by yeast S.c. No. 6 was investigated. It was then confirmed that the appropriate condition was 34 ◦ C, pH 7.0, cell concentration 50 g L−1 (cell dry weight), initial glucose concentration 20 g L−1 , the initial substrate concentration 30 mmol L−1 , cultivation 28 h, the yield was 78.7%, and the enantiomeric excess was 94.4% for (2S,5S)2,5-hexandiol. The experimental results, including the isolation of the special strain with high reduction activity and the optimization of bioreduction conditions presented the foundation for the further scale-up experiment. References Cobley, C.J., Lennon, I.C., Mc, C.R., 2001. On the economic application of DuPHOS rhodium(I) catalysts: a comparison of COD versus NBD precatalysts. Tetrahedron Lett. 42, 7481–7483.