Jeon Eun-Yeong, Baek A-Hyong, Bornscheuer Uwe T, Park Jin-Byung
Department of Food Science & Engineering, Ewha Womans University, Seoul, 120-750, Republic of Korea.
Appl Microbiol Biotechnol. 2015 Aug;99(15):6267-75. doi: 10.1007/s00253-015-6392-9. Epub 2015 Jan 31.
Enzyme fusion was investigated as a strategy to improve productivity of a two-step whole-cell biocatalysis. The biotransformation of long-chain sec-alcohols into esters by an alcohol dehydrogenase (ADH) and Baeyer-Villiger monooxygenases (BVMOs) was used as the model reaction. The recombinant Escherichia coli, expressing the fusion enzymes between the ADH of Micrococcus luteus NCTC2665 and the BVMO of Pseudomonas putida KT2440 or Rhodococcus jostii RHA1, showed significantly greater bioconversion activity with long-chain sec-alcohols (e.g., 12-hydroxyoctadec-9-enoic acid (1a), 13-hydroxyoctadec-9-enoic acid (2a), 14-hydroxyicos-11-enoic acid (4a)) when compared to the recombinant E. coli expressing the ADH and BVMOs independently. For instance, activity of the recombinant E. coli expressing the ADH-Gly-BVMO, in which glycine-rich peptide was used as the linker, with 1a was increased up to 22 μmol g dry cells(-1) min(-1). This value is over 40 % greater than the recombinant E. coli expressing the ADH and BVMO independently. The substantial improvement appeared to be driven by an increase in the functional expression of the BVMOs and/or an increase in mass transport efficiency by localizing two active sites in close proximity.
酶融合作为一种提高两步全细胞生物催化生产力的策略进行了研究。以醇脱氢酶（ADH）和拜耳-维利格单加氧酶（BVMOs）将长链仲醇生物转化为酯作为模型反应。表达藤黄微球菌NCTC2665的ADH与恶臭假单胞菌KT2440或约氏红球菌RHA1的BVMO之间融合酶的重组大肠杆菌，与独立表达ADH和BVMOs的重组大肠杆菌相比，对长链仲醇（如12-羟基十八碳-9-烯酸（1a）、13-羟基十八碳-9-烯酸（2a）、14-羟基二十碳-11-烯酸（4a））表现出显著更高的生物转化活性。例如，表达以富含甘氨酸的肽作为连接子的ADH-Gly-BVMO的重组大肠杆菌对1a的活性提高到22 μmol g干细胞（-1）min（-1）。该值比独立表达ADH和BVMO的重组大肠杆菌高出40%以上。这种显著的提高似乎是由BVMOs功能表达的增加和/或将两个活性位点定位在紧密接近位置导致的传质效率提高所驱动的。
