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Uricase Thermal/pH Stability in Shio-Koji — Computational Analysis (comp-002)

The engineering weakness is loss of assembled, active A. flavus uricase during a multi-day shio-koji process. COMP-002 does not determine whether that happens.

Current evidence boundary

  • Wild-type A. flavus uricase had a reported melting temperature of 27°C and an approximately 38-minute half-life at 40°C in the Imani 2017 study (In Vitro). Those measurements establish thermal sensitivity under their assay conditions; they do not predict retention during a 7–14-day ferment.
  • UniProt and structural records identify Q00511 as a homotetramer (In Vitro structural/biochemical record). Whether the exact produced material remains assembled and active through the intended process is unmeasured.
  • Rezaeian Marjani 2020 reported engineered variants with a higher optimum-activity temperature and longer half-life under thermal stress (In Vitro). The study does not establish the size of a melting-temperature shift or performance in shio-koji.
  • COMP-001 supplies only a fixed-filter and pLDDT-context inventory. Protease survival remains a separate empirical question.

The retired COMP-002 model used an unswept refolding exponent, hard-coded interface weights, arbitrary category boundaries, and pLDDT as a physical interface/integrity proxy. Every quantitative result, failure-mode ranking, and engineering recommendation is invalid. The COMP-002 tombstone is non-runnable; Git retains the retired implementation.

Research conjecture — sub-Tm process attrition may still limit Q00511

Grounded premises: Wild-type A. flavus uricase shows thermal sensitivity in purified assays (In Vitro; Imani 2017, PMID 28667645). The §1.10 process specification defines retention of assembly and activity through the multi-day matrix as a project requirement (Mechanistic Extrapolation). The current corpus contains no direct joint measurement of salt, pH, matrix, proteolysis, and repeated exposure for the exact construct.

Novel leap: Even when the process temperature is below the reported melting temperature, cumulative process exposure might reduce intact tetramer and specific activity enough to become a practical bottleneck. No direct study has tested that multi-factor, multi-day failure mode.

Why it matters: A confirmed thermal/assembly bottleneck would justify variant or process engineering; a negative result would keep attention on other delivery constraints.

Discriminating observation: Under the exact process matrix, measure total protein, intact monomer, tetrameric assembly, and specific activity at day 0, 7, and 14 across the prespecified temperature and pH range.

Experiment that advances or redirects the track

Run validation §1.10 on the exact construct and process. Native-PAGE or another validated assembly readout can distinguish loss of tetramer from loss of abundance; specific activity separates intact protein from functional enzyme. Measure ferment temperature and pH rather than importing a single nominal condition.

If wild type loses assembly or activity reproducibly, compare the exact variant and process arms under the same readouts. A favorable result advances only that configuration. Failure redirects the variant, process, formulation, or delivery track without rejecting uricase or the wider project.

Cross-references