Department of Industrial Engineering, University of Cassino, 03043 Cassino (FR), Italy.
Enzyme Microb Technol. 2011 Apr 7;48(4-5):345-50. doi: 10.1016/j.enzmictec.2010.12.010. Epub 2010 Dec 24.
High yields of nicotinic acid from 3-cyanopyridine bioconversion were obtained by exploiting the in situ nitrile hydratase-amidase enzymatic cascade system of Microbacterium imperiale CBS 498-74. Experiments were carried out in continuously stirred tank UF-membrane bioreactors (CSMRs) arranged in series. This reactor configuration enables both enzymes, involved in the cascade reaction, to work with optimized kinetics, without any purification, exploiting their differing temperature dependences. To this end, the first CSMR, optimized for the properties of the NHase, was operated (i) at low temperature (5°C), limiting inactivation of the more fragile enzyme, nitrile hydratase, (ii) with a high residence time (24 h) to overcome reaction rate limitation. The second CSMR, optimized for the properties of the AMase, was operated (i) at a higher temperature (50°C), (ii) with a lower residence time (6h), and (iii) with a lower substrate (3-cyanopyridine) concentration to control excess substrate inhibition. The appropriate choice of operational conditions enabled total conversion of 3-cyanpyridine (up to 200 mM) into nicotinic acid to be achieved at steady-state and for long periods. Higher substrate concentrations required two CSMRs optimized for the properties of the NHase arranged in series to drive the first reaction to completion.
通过利用Imperiale 微杆菌 CBS 498-74 的原位腈水解酶-酰胺酶酶级联系统，从 3-氰基吡啶生物转化中获得了高产量的烟酸。实验在连续搅拌罐 UF-膜生物反应器（CSMR）中进行，这些 CSMR 串联排列。这种反应器配置可使参与级联反应的两种酶在不进行任何纯化的情况下，利用其不同的温度依赖性，以优化的动力学进行工作。为此，优化了第一个 CSMR 的 NHase 特性（i）在低温（5°C）下操作，以限制更脆弱的酶腈水解酶的失活，（ii）采用高停留时间（24 小时）克服反应速率限制。优化了第二个 CSMR 的 AMase 特性（i）在较高温度（50°C）下操作，（ii）采用较短的停留时间（6 小时），（iii）采用较低的底物（3-氰基吡啶）浓度来控制过量底物抑制。选择适当的操作条件可使 3-氰基吡啶（高达 200mM）在稳态和长时间内完全转化为烟酸。更高的底物浓度需要两个优化了 NHase 特性的 CSMR 串联排列，以推动第一个反应完成。
