Banerjee Anirban, Kaul Praveen, Banerjee U C
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research Sector-67, SAS Nagar, 160 062, Punjab, India.
Appl Microbiol Biotechnol. 2006 Aug;72(1):77. doi: 10.1007/s00253-005-0255-8. Epub 2006 Jan 4.
(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (phi). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.
使用含有立体选择性腈水解酶的恶臭假单胞菌MTCC 5110的游离细胞和固定化细胞，从外消旋扁桃腈生产(R)-扁桃酸。除了优化培养条件和培养基成分外，还建议采用诱导剂补料方法来提高酶产量，从而提高扁桃腈的转化率。还研究了细胞生长周期与酶积累之间的关系，并将诱导剂的添加延迟6小时以实现最大腈水解酶活性。通过十二烷基磷酸钠-聚丙烯酰胺凝胶电泳分析也验证了腈水解酶的表达。恶臭假单胞菌MTCC 5110细胞进一步固定在海藻酸钙中，并将固定化生物催化剂制剂用于扁桃腈的对映选择性水解。基于Thiele模数(φ)对固定化系统进行了表征。由于固定化，实现了高效的生物催化剂循环利用，即使在20次分批循环后，固定化细胞仍表现出88%的转化率。最后，建立了一个制备规模的补料分批反应，以生产1.95 g对映体过量为98.8%的(R)-(-)-扁桃酸。
