在含有固定化多酶系统和葡聚糖-NAD+的人工细胞的生物反应器中生产必需的L-支链氨基酸。

Production of essential L-branched-chain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+.

作者信息

Gu K F, Chang T M

机构信息

Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada.

出版信息

Biotechnol Bioeng. 1990 Jul;36(3):263-9. doi: 10.1002/bit.260360308.

DOI:10.1002/bit.260360308

PMID:18595077

Abstract

We prepared artificial cells each containing leucine dehydrogenase (EC 1.4.1.9), urease (EC 3.5.1.5), soluble dextran-NAD(+), and one of the following coenzyme regenerating dehydrogenases: glucose dehydrogenase (EC 1.1.1.47); yeast alcohol dehydrogenase (EC 1.1.1.1); malate dehydrogenase (EC 1.1.1.37); or lactate dehydrogenase (EC 1.1.1.27). Artificial cells were packed in small columns. L-Leucine, L-valine, and L-isoleucine were continuously produced with simultaneous dextran-NADH regeneration. The maximum production ratios depended on the coenzyme regenerating systems used: 83-93% for D-glucose and glucose dehydrogenase system; 90% for ethanol and yeast alcohol dehydrogenase system; 45-55% for L-malate and malate dehydrogenase system; and 64-78% for L-lactate and lactate dehydrogenase system. Kinetic experiments were also carried out. The apparent K(m) values are as follows: 0.33 mM for alpha-ketoisocaproate (KIC); 0.51 mM for alpha-ketoisovalerate (KIV); 0.58 mM for DL-alpha-keto-beta-methyl-n-valerate (KMV); 3.52 mM for urea; 27.82 mM for D-glucose; 3.89 mM for ethanol; 3.02 mM for L-malate; and 16.67 mM for L-lactate. Kinetic analysis showed that KIC, KIV, and KMV were all competitive inhibitors in the reactions catalyzed by leucine dehydrogenase. Their inhibitor constants were the corresponding K(m) values.

摘要

我们制备了人工细胞，每个细胞都含有亮氨酸脱氢酶（EC 1.4.1.9）、脲酶（EC 3.5.1.5）、可溶性葡聚糖 - NAD(+)以及以下辅酶再生脱氢酶之一：葡萄糖脱氢酶（EC 1.1.1.47）；酵母乙醇脱氢酶（EC 1.1.1.1）；苹果酸脱氢酶（EC 1.1.1.37）；或乳酸脱氢酶（EC 1.1.1.27）。人工细胞被填充到小柱中。L - 亮氨酸、L - 缬氨酸和L - 异亮氨酸在葡聚糖 - NADH再生的同时持续产生。最大产率取决于所使用的辅酶再生系统：D - 葡萄糖和葡萄糖脱氢酶系统为83 - 93%；乙醇和酵母乙醇脱氢酶系统为90%；L - 苹果酸和苹果酸脱氢酶系统为45 - 55%；L - 乳酸和乳酸脱氢酶系统为64 - 78%。还进行了动力学实验。表观K(m)值如下：α - 酮异己酸（KIC）为0.33 mM；α - 酮异戊酸（KIV）为0.51 mM；DL - α - 酮 - β - 甲基 - n - 戊酸（KMV）为0.58 mM；尿素为3.52 mM；D - 葡萄糖为27.82 mM；乙醇为3.89 mM；L - 苹果酸为3.02 mM；L - 乳酸为16.67 mM。动力学分析表明，KIC、KIV和KMV在亮氨酸脱氢酶催化的反应中均为竞争性抑制剂。它们的抑制常数即为相应的K(m)值。