期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

毕赤酵母表达重组猪肾氨基酰化酶Ⅰ的纯化与鉴定 被引量：1

Purification and Characterization of Recombinant Porcine Aminoacylase Ⅰ Expression in Pichia pastoris
下载PDF
导出
摘要 运用毕赤氏酵母对猪源重组氨基酰化酶Ⅰ(pACY1)进行了表达,并对其酶学参数进行了测定。构建毕赤氏酵母工程菌GS115-pacy1,通过甲醇诱导表达后,进行了Phenyl-Sepharose及Q-Sepharose两步色谱纯化;对纯化后重组pACY1的动力学参数进行测定。重组pACY1纯化后呈均一性,其纯度为98.6%;酶学参数测定结果表明,重组酶的比活力(Vmax)为280U/mg,米氏常数(Km)为0.91mM,接近天然酶对应的287U/mg和0.84mM;而酵母重组酶Tm值为74℃,高于天然酶的70℃。毕赤氏酵母表达重组pACY1的酶活性与天然酶相当,且其热稳定性优于天然酶。这为氨基酸拆分产业提供了更加优质而丰富的酶制剂。 Recombinant porcine Aminoacylase Ⅰ （pACY1） was expressed in Pichia pastoris, and had been charac-tered. The recombinant strain GS115-pacyl was constructed. Recombinant pACY1 were purified from its culture after methanol inducing by Phenyl-Sepharose and Q-Sepharose. Then the kinetic parameters of the purified enzymes were determined. The purified enzyme was homogenicity. Ⅰts purity was 98.6%. Specific activity （Vmax） and Michaelis eonstant（Km） of it were 280U/mg and 0.91mM, similar to 287U/mg and 0.84mM of natural porcine enzyme individ-ually. The Tm value of recombinant pACY1 was 74℃, higher than 70℃ of natural enzyme. The activity of recombi-nant enzyme expressed in Pichia pastoris was similar with natural enzyme, but the heat stablity of former was better than the latter＇s. Ⅰt will provide abundant and high-grade enzyme for chiral aimino acid industry.
作者 刘志刚 杨波 胡征 邓贝
机构地区 湖北工业大学生物工程学院
出处 《食品与发酵科技》 CAS 2012年第4期95-99,共5页 Food and Fermentation Science & Technology
基金 湖北省教育厅基金项目(Q20081411) 湖北工业大学高层次人才基金项目(20062016)的资助
关键词 氨基酰化酶Ⅰ 毕赤氏酵母 表达纯化 热稳定性 minoacylase Ⅰ Pichia pastoris expression and purification heat stability
分类号 TQ926 [轻工技术与工程—发酵工程]
  • 相关文献

参考文献13

  • 1Yokorgawa K, Sato E, Esaki N, et al. Enantioselec- tire synthesis of N-acetyl-L-methionine with aminoa- cylase in organic solvent[J]. Appl Microbiol Biotech- nol, 1994(42) : 287-289.
  • 2Tsunasawa S. Does uncontrolled survival of acetylated peptides lead to cell proliferation? Deletion of N-ter- minal deacetylating system for protein/peptide in small lung carcinoma cells [J]. J. Lab. Clin. Med,1992 (120) :505-506.
  • 3Scaloni A, Jones W M,Pospischil M,et al. Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines[J]. J. Lab. Clin. Med,1992(120):546-552.
  • 4Mitta M,Kato I,Tsunasawa S. The mucleotide sequence of human aminoacylase-l[J]. Biochimica et Biophysi- ca Acta, 1993(1174) :201-203.
  • 5Maceyka M, Nava V E, Milstien S, et al. Aminoacy- lase 1 is a sphingosine kinase 1-interacting protein [J]. FEBS Letters,2004(568):30-34.
  • 6Pittelkow S, Lindner H, Rhm K H. Human and porcine aminoacylase 1 overproduced in a baculovirus expression vector system:evidence for structural and functional identity with enzymes isolated from kidney[J]. Protein Expr. Purif, 1998 ( 12 ) : 269-276.
  • 7Liu Z, Zhen Z, Li W, et al. Probing the catalytic center of porcine aminoacylase 1 by site-direeted mu- tagenesis,homology modeling and substrate docking[J]. J. Biochem,2006(139) :421-430.
  • 8Wardenga R,Hollmann F,Bomscheuer U, et al. Func- tional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones [J]. Appl. Microbiol. Biotechnol,2008 (81) : 721-729.
  • 9Wardenga R, Lindner H, Hollmann F, et al. Increas- ing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mntagenesis [J]. Biochimie,2010 (92): 102-109.
  • 10Lindner H, Berens W, R6hm K H, et al. Mutational analysis of two PWW sequence motifs in human aminoacylase 1 [J]. Biol. Chem,2000(381) : 1055-1061.

二级参考文献6

同被引文献20

  • 1Mitta M,Kato I,Tsunasawa S. The mucleotide sequence of human aminoacylase-1[J].{H}Biochimica et Biophysica Acta,1993,(1174):201-203.
  • 2Tsunasawa S. Does uncontrolled survival of acetylated peptides lead to cell proliferation? Deletion of N-ter-minal deacetylating system for protein/peptide in small lung carcinoma cells[J].{H}Journal of Laboratory and Clinical Medicine,1992,(120):505-506.
  • 3Scaloni A,Jones W M,Pospischil M. Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines[J].{H}Journal of Laboratory and Clinical Medicine,1992,(120):546-552.
  • 4Maceyka M,Nava V E,Milstien S. Aminoacylase 1 is a sphingosine kinase 1-interacting protein[J].{H}FEBS Letters,2004,(568):30-34.
  • 5Sass J O,Olbrich H,Mohr V O. Neurological findings in aminoacylase 1 deficiency[J].{H}NEUROLOGY,2007,(68):2151-2153.
  • 6Tylki-Szymanska A,Gradowska W,Sommer A. Aminoacylase 1 deficiency associated with autistic be-havior[J].{H}Journal of Inherited Metabolic Disease,2010.
  • 7Sommer A,Christensen E,Schwenger S. The molecular basis of aminoacylase 1 deficiency[J].{H}Biochimica et Biophysica Acta,2011,(1812):685-690.
  • 8Henseling J,R?hm K H. Aminoacylase I from hog kidney:anion effects and the pH dependence of ki-netic parameters[J].{H}Biochimica et Biophysica Acta,1988,(959):370-377.
  • 9Denton P. A structure-activity relationship for the hy-drolysis of acetylamino acids by porcine aminoacylase[J].{H}Bioorganic Chemistry,2000,(28):205-213.
  • 10Heese D,Berger S,R?hm K H. Nuclear magnetic relaxation studies of the role of the metal ion in Mn2+-subtituted aminoacylase 1[J].{H}European Journal of Biochemistry,1990,(188):175-180.

引证文献1

食品与发酵科技
2012年 第4期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部