报告题目：Microbial nitrile metabolism: from biochemical and genetic studies to industrial application
报 告 人：日本筑波大学 小林达彦教授
Nitrile compounds are widely manufactured and extensively used by the chemical industry. Nitrile herbicides are also widely used in agriculture. These nitriles have been widely spread out in our environment in forms of industrial waste water and residual agricultural chemicals, and if their release is not controlled, finally they will threaten the environment.
We have studied microbial degradation of highly toxic nitriles (R-CN) containing a cyano functional group. The microbial degradation of nitriles has been found to proceed through two enzymatic pathways; nitrile hydratase (NHase) catalyzes the hydration of a nitrile to the corresponding amide (R-CONH2) followed by its conversion to the acid (R-COOH) plus ammonia by amidase, whereas nitrilase catalyzes the direct hydrolysis of a nitrile to the corresponding acid plus ammonia. We have characterized both enzymes and their genes from several microorganisms. Particularly, we have focused on an actinomycete Rhodococcus rhodochrous J1. Although this strain produces only nitrilase when isovaleronitrile is added into the culture medium as an inducer, it also produces two NHases depending on an inducer in the presence of cobalt ions; when the strain is cultured in the medium containing urea and cyclohexanecarboxamide, high-molecular-mass- (H-) and low-molecular-mass- (L-) NHases are selectively induced, respectively.The industrial production of acrylamide from acrylonitrile by H-NHase is under way. We clarified the transcriptional regulation mechanisms of the both NHase gene and nitrilase gene of R. rhodochrous J1. These strong induction systems are expected to be applicable for constructing regulatory expression systems for actinomytcetes. We investigated whether both Rhodococcus regulatory systems function in Streptomyces and we finally constructed an expression vector pSH19 containing a novel regulatory expression system (PnitA-NitR system), which is based on the nitrilase regulation mechanims. Heterologous protein expression experiments for evaluation of the functionality and ability of pSH19 revealed that the PnitA-NitR system is strongly induced, and is able to express a target protein as high as ~ 40% of all soluble protein in Streptomyces.
We also have studied nitrile metabolism in another industrial strain, Pseudomonas chlororaphis B23. We clarified new functions of enzymes involved in the microbial metabolism.