Combinatorial approach for screening and assessment of multiple therapeutic enzymes from marine isolate<i>Pseudomonas aeruginosa</i>AR01

Jagadeesan, Yogeswaran; Meenakshisundaram, Shanmugapriya; Boopathy, Lokha Ranjani Alagar; Mookandi, Vijay Pradhap Singh; Balaiah, Anandaraj

doi:10.1039/c9ra02555c

Cited by 5 publications

(2 citation statements)

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“…In this work, we evaluated two quantitative methods to develop a high-throughput screening approach of bacterial uricase activity in microtiter plates, with particular focus on conditions relevant to lactobacilli, Bacillus , and Bifidobacterium strains. Using a stepwise approach, we aimed at developing a sensitive, yet robust method using whole bacterial suspensions to enable the simultaneous detection of both extracellular and cell-associated uricase activities, thereby accelerating the screening of large microbial collections and the identification of uricolytic strains independently from the enzyme’s partition ( Watanabe et al, 1969 ; Olivieri et al, 1983 ; Suzuki et al, 2004 ; Kai et al, 2008 ; Lotfy, 2008 ; Zhang et al, 2010 ; Khucharoenphaisan and Sinma, 2011 ; Handayani et al, 2018 ; Jagadeesan et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

“…To date, uricase activity was biochemically confirmed in various bacterial taxa, either extracellularly in Pseudomonas strains ( Abdel-Fattah et al, 2005 ; Jagadeesan et al, 2019 ), or intracellularly in Arthrobacter ( Suzuki et al, 2004 ), Microbacterium ( Zhou et al, 2005 ; Kai et al, 2008 ), Streptomyces ( Watanabe et al, 1969 ), Saccharopolyspora ( Khucharoenphaisan and Sinma, 2011 ), Micrococcus ( Olivieri et al, 1983 ), Metabacillus ( Zhang et al, 2010 ), and Bacillus ( Mahler, 1970 ; Bongaerts and Vogels, 1976 ; Bongaerts et al, 1978 ; Huang and Wu, 2004 ; Lee et al, 2005 ; Lotfy, 2008 ) strains. In few lactobacilli strains, uricase activity was suggested to be present both intracellularly and extracellularly ( Handayani et al, 2018 ), thus prompting further investigation of uricase in taxa associated with probiotic strains.…”

Section: Introductionmentioning

confidence: 99%

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Optimized UV-Spectrophotometric Assay to Screen Bacterial Uricase Activity Using Whole Cell Suspension

et al. 2022

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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 H2O2-based fluorescent assays to enable the rapid identification of uricase activity in a broad panel of lactobacilli, Bacillus, and Bifidobacterium species. We highlighted abiotic (medium composition and mode of sterilization) and biotic (H2O2-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 via liquid chromatography. Finally, a panel of 319 Qualified Presumption of Safety (QPS) strains of lactobacilli (18 species covering nine genera), Bacillus (three species), and Bifidobacterium (four species) were screened for uricase activity using the optimized method. All 319 strains, but the positive control Bacillus 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.

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