PSI-6 Characterization of in vitro stability for two multi-functional processive endoglucanases as exogenous biocatalysts in pig nutrition
Bibliographic record
Abstract
Abstract Poor efficiency of dietary fibre utilization limits global pork production profit margins and mitigation of footprint on environment. The objective of this study was to characterize in vitro stability for two unique processive endoglucanases of tCel5A1 and p4818Cel5_2A that are reported to hydrolyze natural cellulose and have multi-functionality towards hemicelluloses (Basit and Akhtar, Biotechnology and Bioengineering, 2018, 115:1675; Wang et al., Scientific Reports 2019, 9:13630). Both tCel5A1 and p4818Cel5_2A were modelled with the SWISS-MODEL online server and analyzed and visualized by PyMOL2.4.1. These two cellulases were overexpressed in ClearColi®BL21(DE3). Their endoglucanase activities were determined using 0.70 % sodium carboxymethyl cellulose and 5 mM dithiothreitol (DTT) with or without N2 gas purging; and incubated at pH 6.5–7.0 and 37 °C for 15 min. The three-dimensional models showed 1 and 4 Cys residues on the surfaces of tCel5A1 and p4818Cel5_2A, respectively, suggesting their susceptibility to auto-oxidation by air-borne O2. This prediction was tested by comparing the activities of N2-purged and non-purged enzyme preparations as well as their processing and incubation handling. Both tCel5A1 and p4818Cel5_2A activities were enhanced (P < 0.01) by 50% when 5-mM DTT and N2-purging were adopted. Furthermore, after incubating both tCel5A1 and p4818Cel5_2A enzyme preparations under the porcine gastric pH (3.5) and pepsin (274 U/mL) as well as intestinal trypsin (78 U/mL) and chymotrypsin (20 U/mL) activities at pH 6.5 during 0–5 h, Eadie-Hofstee inhibition kinetic analyses showed that tCel5A1 and p4818Cel5_2A respectively lost 18 and 68% (P < 0.01) of their initial activities after 2 h under the gastric conditions and more than 90% (P < 0.01) of their initial activities after 2–3 h under the intestinal conditions. Therefore, further enzyme protein engineering and/or post-fermentation treatments, such as coating for by-passing the gastric-intestinal environment, will be required to enable these two processive endoglucanases as efficacious exogenous fibre enzymes.
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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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 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 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".