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 syn- thesis, 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 （asnA） from Escherichia coli （E. coli） JM109 was amplified and transformed into E. coli CICC 11022S using molec- ular biological methods, and then the ASNA was expressed. The molecular weight of expressed protein was of about 37 kDa 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 l1022S/pET28a （ ＋ ）-asnA 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）.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2016年第8期57-60,共4页
Food and Fermentation Industries
关键词
一锅法
富马酸
天冬酰胺合成酶
L-天冬酰胺
生物转化
one-pot reaction
fumaric acid
Asparagine synthetase A
L-asparagine
biotransformation
