“一锅法”生物转化富马酸制备L-天冬酰胺
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摘要
L-天冬酰胺广泛应用于食品、医药、化工合成和微生物培养等领域。目前工业上主要依靠化学合成和直接提取方法制备。该研究首次采用双酶催化富马酸"一锅法"制备L-天冬酰胺。克隆来源于大肠埃希氏菌JM109的天冬酰胺合成酶A基因asn A,并于产L-天冬氨酸酶的E.coli CICC 11022S中表达,表达的蛋白分子质量约为37 k Da,与预期大小相符,比酶活力为1443.7 U/g。利用构建的工程菌E.coli CICC 11022S/p ET28a(+)-asn A全细胞高密度催化富马酸生产L-天冬酰胺,以高效液相色谱法及PITC柱前衍生-高效液相法检测底物、中间产物和终产物,转化10 h,富马酸转化率为94.7%,L-天冬酰胺产量可达125.1g/L,生产速率为12.51 g/(L·h)。
L-asparagine was one of natural amino acids,which was widely used in food,medicine,chemical synthesis,microbial culture and so on,and mainly obtained by chemical synthesis or direct extraction methods. In this study,one-pot reaction was reported to synthesize L-asparagine by two kinds of enzyme. Asparagine synthetase gene A( asn A) from Escherichia coli( E. coli) JM109 was amplified and transformed into E. coli CICC 11022 S using molecular biological methods,and then the ASNA was expressed. The molecular weight of expressed protein was of about37 k Da by SDS-PAGE,which was consistent with the expected size. After analyzing the ASNA activity,the enzyme activity was 1 443. 7 U / g wet cell. Finally,the constructed strains E. coli CICC 11022 S / p ET28a( +)-asn A was applied on conversion from fumaric acid to L-asparagine. After catalyzing for 10 h,the substrate and product were detected by PITC column derivatization-high performance liquid. The conversion ratio of fumaric acid was 94. 7%,the yield of L-asparagine was up to 125. 1 g / L,and the production rate was 12. 51 g /( L·h).
L-asparagine was one of natural amino acids,which was widely used in food,medicine,chemical synthesis,microbial culture and so on,and mainly obtained by chemical synthesis or direct extraction methods. In this study,one-pot reaction was reported to synthesize L-asparagine by two kinds of enzyme. Asparagine synthetase gene A( asn A) from Escherichia coli( E. coli) JM109 was amplified and transformed into E. coli CICC 11022 S using molecular biological methods,and then the ASNA was expressed. The molecular weight of expressed protein was of about37 k Da by SDS-PAGE,which was consistent with the expected size. After analyzing the ASNA activity,the enzyme activity was 1 443. 7 U / g wet cell. Finally,the constructed strains E. coli CICC 11022 S / p ET28a( +)-asn A was applied on conversion from fumaric acid to L-asparagine. After catalyzing for 10 h,the substrate and product were detected by PITC column derivatization-high performance liquid. The conversion ratio of fumaric acid was 94. 7%,the yield of L-asparagine was up to 125. 1 g / L,and the production rate was 12. 51 g /( L·h).
引文
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[11]MANHAS R,TRIPATHI P,KHAN S,et al.Identification and functional characterization of a novel bacterial type asparagine synthetase A:a tRNA synthetase paralog from Leishmania donovani[J].Journal of Biological Chemistry,2014,289(17):12 096-12 108.
[12]张奇,张露,徐友强,等.生物转化法制备L-天冬酰胺[J].中国生物工程杂志,2016,36(1):63~67.
[13]LI L,LIU Y H,WAN Y,et al.Efficient enzymatic synthesis of guanosine 5’-diphosphate-sugars and derivatives[J].Organic Letters,2013,15(21):5 528-5530.
[2]张维燕,刘亚亚,刘旭平,等.谷氨酰胺和天冬酰胺对CHO细胞生长、代谢及抗体表达的影响[J].中国生物工程杂志,2014,34(4):9-15.
[3]ZHANG J,FAN J,VENNETI S,et al.Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion[J].Molecular Cell,2014,56(2):205-218.
[4]BURDA P,AEBI M.The dolichol pathway of N-linked glycosylation[J].Biochimica et Biophysica Acta,1999,1 426(2):239-257.
[5]IMPERIALI B,O'CONNOR S E.Effect of N-linked glycosylation on glycopeptide and glycoprotein structure[J].Current Opinion in Chemical Biology,1999,3(6):643-649.
[6]PATTERSON M C.Metabolic mimics:the disorders of Nlinked glycosylation[J].Seminars in Pediatric Neurology,2005,12(3):144-151.
[7]詹谷宇,叶明,田萍.从草木樨芽提取天门冬酰胺[J].氨基酸和生物资源,1984(2):6-8.
[8]黄宜基,周承文,翟元凤,等.L-天门冬氨酸胺一水合物的合成[J].氨基酸和生物资源,1984(2):4-5.
[9]RICHARDS N G,SCHUSTER S M.Mechanistic issues in asparagine synthetase catalysis[J].Advances in Enzymology and Related Areas of Molecular Biology,1998,72(1):145-98.
[10]OHASHI M,ISHIYAMA K,KOJIMA S,et al.Asparagine synthetase1,but not asparagine synthetase2,is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots[J].Plant and Cell Physiology,2015,56(4):769-778.
[11]MANHAS R,TRIPATHI P,KHAN S,et al.Identification and functional characterization of a novel bacterial type asparagine synthetase A:a tRNA synthetase paralog from Leishmania donovani[J].Journal of Biological Chemistry,2014,289(17):12 096-12 108.
[12]张奇,张露,徐友强,等.生物转化法制备L-天冬酰胺[J].中国生物工程杂志,2016,36(1):63~67.
[13]LI L,LIU Y H,WAN Y,et al.Efficient enzymatic synthesis of guanosine 5’-diphosphate-sugars and derivatives[J].Organic Letters,2013,15(21):5 528-5530.