Improvement in the Thermal Stability of Pyrophosphatase by Conjugation to Poly(<i>N</i>-isopropylacrylamide): Application to the Polymerase Chain Reaction
Bibliographic record
Abstract
Polymerase chain reaction (PCR) is a powerful method for nucleic acid amplification. However, the PCR is inhibited in its yield due to its byproduct, pyrophosphate (PPi), a byproduct of the reaction; the yield is thereby limited. The conventional method for hydrolysis of PPi by pyrophosphatase (PPase) is not well adapted for operation at elevated temperatures over long times as required during the PCR. In this work, we reported a strategy to improve the PCR yield using a conjugate of the enzyme with the thermally responsive polymer poly(N-isopropylacrylamide) (PNIPAM). Pyrophosphatase (PPase) was conjugated to PNIPAM site-specifically near the active center. As compared to the free enzyme, the optimum temperature of the conjugate was shown to increase from 45 to 60 °C. For the conjugate, about 77% enzyme activity was retained after incubation at 60 °C for 3 h, representing a 6.8-fold increase as compared to the unconjugated enzyme. For the PCR using the conjugate, the yield was 1.5-fold greater than using the unconjugated enzyme. As well as improving the yield of the PCR (and possibly other biological reactions) at elevated temperature, polymer conjugation may also provide a strategy to improve the heat resistance of proteins more generally.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 teacher head, 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".