Characterization and overproduction of the <i>Escherichia coli</i> <i>appA</i> encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities
Bibliographic record
Abstract
The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44,708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60 degrees C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K(m) and Vmax values of 0.79 mM and 3165 U.mg-1 of protein for phytase activity and 5.5 mM and 712 U.mg-1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K(m) and Vmax values. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U.mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".