A Practical System for High-Throughput Screening of Mutants of Bacillus fastidiosus Uricase

Abstract

For high-throughput screening (HTS) of Bacillus fastidiosus uricase mutants, a practical system was proposed. By error-prone PCR with final 1.5 mM MnCl, two focused libraries of mutants for A1-V158 and V150-D212 were generated separately. After induced expression of individual clones in 48-well microplates, Escherichia coli cells (BL21) were lyzed by 1.0 M Tris-HCl at pH 9.0 in 96-well microplates at 25 °C for 7.5 ~ 10.5 h; uricase reaction was continuously monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm to estimate V /K by kinetic analysis of reaction curve for comparison. V /K was resistant to initial uric acid levels with an upper limit 3-fold over that of initial rates. By receiver-operator-characteristic analysis of the recognition of the one of higher activity in uricase pair whose specific activity ratio was 1.8 or 3.3, the area-under-the-curve was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was thus developed to recognize positive mutants of 1-fold higher activity. Indeed, mutant L171I/Y182F/Y187F/A193S of higher activity but lower thermostability at pH 7.4 and mutant V144A of higher activity and consistent thermostability were discovered. Therefore, the proposed system was practical for HTS of uricase mutants.

Catalytic Mechanisms for Cofactor-Free Oxidase-Catalyzed Reactions: Reaction Pathways of Uricase-Catalyzed Oxidation and Hydration of Uric Acid

“…25 Briefly, the pET28a vector was used for Bacillus fastidiosus uricase and its mutants including Asn271Leu or Gln299Ile mutation (generated by site-directed mutagenesis). The proteins were expressed in Escherichia coli BL21 (DE3), and cells collected for each protein (wild-type or mutant) were broken in a lysis buffer (20 mM Tris-HCl at pH 8.0) by sonication treatment (30% amplitude, 4-s treatment at 5-s intervals).…”

“…The experimental methods used in this study are similar to what we previously described . Briefly, the pET28a vector was used for Bacillus fastidiosus uricase and its mutants including Asn271Leu or Gln299Ile mutation (generated by site-directed mutagenesis).…”

Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

“…With PAAS/mutants, 2.0 mM potassium 4-nitrophenylsulfate was used in 1.0 mM Tris–HCl at pH 9.0 to measure the absorbance at 405 nm with Biotek ELX 800 [1] , [3] . With BFU/mutants, 0.14 mM uric acid was employed in 0.20 M sodium borate at pH 9.2 to measure absorbance at 293 nm with Biotek EON [1] , [4] . Their initial rates were derived from absorbance change from 10.0 to 15.0 min since reaction initiation [1] .…”

Metabacillus kandeliae sp. nov., isolated from the rhizosphere soil of a decayed mangrove plant Kandelia candel

“…Many studies confirmed that these strains had strong biological significance; for example, fermentation extract of Sargassum horneri by Metabacillus litoralis M3 (SFE-12) promotes plant growth and xanthorhamnin from Metabacillus indicus TMC-6 significantly contributes to UV-induced radiation oxidative stress resistance [12, 13]. In addition, the uricase of Metabacillus fastidiosus has been studied by Bongaerts et al [14, 15].…”