Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 84538 Bratislava, Slovakia.
Biosens Bioelectron. 2013 Dec 15;50:235-8. doi: 10.1016/j.bios.2013.06.061. Epub 2013 Jul 8.
A whole-cell amperometric biosensor consisting of genetically engineered Escherichia coli immobilised in polyelectrolyte membrane onto a miniaturised oxygen electrode was developed and used for monitoring of biotransformation based on Baeyer-Villiger oxidation. Baeyer-Villiger oxidation is commonly performed using microorganisms overexpressing Baeyer-Villiger monooxygenase enabling the production of enantiopure lactones or esters used in pharmaceutical industry. The biorecognition element, genetically modified E. coli overexpressing either cyclopentanone monooxygenase or cyclohexanone monooxygenase was immobilised in the form of solid polyelectrolyte complex gel membrane made of cellulose sulphate, sodium alginate and poly(methylene-co-guanidine) and attached to the surface of miniaturised oxygen electrode. The time response of the biosensor was 30s, the linear range of the calibration curve (R(2)=0.9993) was 8-130 μM and the sensitivity was 1.8 nA μM(-1) (RSD=5.0%) for substrate of Baeyer-Villiger oxidation (±)-cis-bicyclo[3.2.0]hept-2-en-6-one as analyte. The biosensor sensitivity was assessed for two other commercially available substrates, 4-methylcyclohexanone and 3-methylcyclohexanone. No interferences from ampicillin, citric acid, acetic acid, ethanol, methanol, glucose and products of Baeyer-Villiger oxidation (1R, 5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one and (1S, 5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one were detected. After 1 week of storage at 4°C the biosensor sensitivity was without changes. The biosensor was employed for monitoring of Baeyer-Villiger biotransformation and the results were correlated with gas chromatography. Till now, this is the first described biosensor based on Baeyer-Villiger monooxygenase and the first reported application of biosensor for monitoring of biotransformation based on Baeyer-Villiger oxidation.
一种全细胞安培生物传感器，由基因工程大肠杆菌固定在聚电解质膜上，然后固定在微型氧电极上，用于监测基于 Baeyer-Villiger 氧化的生物转化。Baeyer-Villiger 氧化通常使用过表达 Baeyer-Villiger 单加氧酶的微生物进行，从而生产用于制药行业的手性纯内酯或酯。生物识别元件是基因修饰的大肠杆菌，过表达环戊酮单加氧酶或环己酮单加氧酶，以纤维素硫酸盐、海藻酸钠和聚（亚甲基-co-胍）的固体聚电解质复合物凝胶膜的形式固定，并附着在微型氧电极的表面上。生物传感器的时间响应为 30s，校准曲线的线性范围（R²=0.9993）为 8-130 μM，灵敏度为 1.8 nA μM⁻¹（RSD=5.0%），用于 Baeyer-Villiger 氧化的底物（±）-顺式-双环[3.2.0]庚-2-烯-6-酮作为分析物。还评估了该生物传感器对另外两种市售底物 4-甲基环己酮和 3-甲基环己酮的灵敏度。没有检测到氨苄西林、柠檬酸、乙酸、乙醇、甲醇、葡萄糖和 Baeyer-Villiger 氧化产物（1R，5S）-3-氧杂双环[3.3.0]辛-6-烯-2-酮和（1S，5R）-2-氧杂双环[3.3.0]辛-6-烯-3-酮的干扰。在 4°C 下储存 1 周后，生物传感器的灵敏度没有变化。该生物传感器用于监测 Baeyer-Villiger 生物转化，结果与气相色谱相关联。到目前为止，这是第一个基于 Baeyer-Villiger 单加氧酶的生物传感器，也是第一个报道的用于监测基于 Baeyer-Villiger 氧化的生物转化的生物传感器。
