Pugin Benoit, Plüss Serafina, Mujezinovic Denisa, Nielsen Rikke C, Lacroix Christophe
Laboratory of Food Biotechnology, Department of Health Sciences and Technology, ETH Zürich, Zürich, Switzerland.
Beo Therapeutics AG, Zürich, Switzerland.
Front Microbiol. 2022 Apr 13;13:853735. doi: 10.3389/fmicb.2022.853735. eCollection 2022.
Uricase catalyzes the conversion of uric acid into allantoin with concomitant reduction of molecular oxygen to hydrogen peroxide. In humans, uricase is not functional, thereby predisposing individuals to hyperuricemia, a metabolic disturbance associated with gout, chronic kidney disorders, and cardiovascular diseases. The efficacy of current therapies to treat hyperuricemia is limited, and novel approaches are therefore desired, for instance using uricase-expressing probiotic strains. Here, we evaluated UV-spectrophotometric and HO-based fluorescent assays to enable the rapid identification of uricase activity in a broad panel of lactobacilli, , and species. We highlighted abiotic (medium composition and mode of sterilization) and biotic (HO-producing strains) factors impacting the measurements' accuracy, and reported on the stepwise optimization of a simple, fast, and robust high-throughput UV-spectrophotometric method to screen uricase activity using whole bacterial suspension, thereby assessing both cell-associated and extracellular activity. The validity of the optimized assay, based on the monitoring of uric acid degradation at 300 nm, was confirmed liquid chromatography. Finally, a panel of 319 Qualified Presumption of Safety (QPS) strains of lactobacilli (18 species covering nine genera), (three species), and (four species) were screened for uricase activity using the optimized method. All 319 strains, but the positive control sp. DSM 1306, were uricase-negative, indicating that this activity is rare among these genera, especially in isolates from food or feces. Altogether, the UV-spectrophotometric high-throughput assay based on whole bacterial suspension reported here can be used to rapidly screen large microbial collections, by simultaneously detecting cell-associated and extracellular uricase activity, thereby accelerating the identification of uricolytic strains with therapeutic potential to treat hyperuricemia.
尿酸酶催化尿酸转化为尿囊素，同时将分子氧还原为过氧化氢。在人类中，尿酸酶没有功能，因此使个体易患高尿酸血症，这是一种与痛风、慢性肾脏疾病和心血管疾病相关的代谢紊乱。目前治疗高尿酸血症的疗法效果有限，因此需要新的方法，例如使用表达尿酸酶的益生菌菌株。在这里，我们评估了紫外分光光度法和基于过氧化氢的荧光测定法，以便能够在广泛的乳酸杆菌属、双歧杆菌属和嗜热栖热菌属物种中快速鉴定尿酸酶活性。我们强调了影响测量准确性的非生物因素（培养基组成和灭菌方式）和生物因素（产生过氧化氢的菌株），并报告了一种简单、快速且稳健的高通量紫外分光光度法的逐步优化过程，该方法使用全细菌悬浮液筛选尿酸酶活性，从而评估细胞相关和细胞外活性。基于在300 nm处监测尿酸降解的优化测定法的有效性通过液相色谱法得到了证实。最后，使用优化方法对一组319株合格假定安全（QPS）的乳酸杆菌（涵盖九个属的18个物种）、双歧杆菌属（三个物种）和嗜热栖热菌属（四个物种）菌株进行了尿酸酶活性筛选。除阳性对照嗜热栖热菌DSM 1306外，所有319株菌株均为尿酸酶阴性，这表明该活性在这些属中很少见，尤其是在食品或粪便分离株中。总之，本文报道的基于全细菌悬浮液的紫外分光光度高通量测定法可用于快速筛选大量微生物集合，通过同时检测细胞相关和细胞外尿酸酶活性，从而加速鉴定具有治疗高尿酸血症潜力的尿酸分解菌株。
