The Impact of Streptomyces griseus Protease Reserved for Protein Evaluation of Ruminant Feed on Carbohydrase Activity during Co-Incubation
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
- to determine whether the S. griseus protease mixture is active at 39 °C and pH 6.75, measuring the hydrolysis of two peptides through an increased fluorescence signal (fluorescence assay) and,
- to investigate the relationship between increasing doses of S. griseus protease mixture on the release of reducing sugars due to the degradation of starch by α-amylase (absorbance assay).
2.1. Enzymes and Buffer Solution
2.2. Substrates
2.3. Fluorescence Assay
2.4. Absorbance Assay
2.5. Statistical Analysis
3. Results
3.1. Preliminary Experiments
3.2. Main Experiments
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Licitra, G.; Lauria, F.; Carpino, S.; Schadt, I.; Sniffen, C.J.; Van Soest, P.J. Improvement of the Streptomyces Griseus Method for Degradable Protein in Ruminant Feeds. Anim. Feed Sci. Technol. 1998, 72, 1–10. [Google Scholar] [CrossRef]
- Nomoto, M.; Narahashi, Y.; Murakami, M. A Proteolytic Enzyme of Streptomyces Griseus VI. Hydrolysis of Protein by Streptomyces Griseus. J. Biochem. 1960, 48, 593–602. [Google Scholar] [CrossRef]
- Okon, P.; Bachmann, M.; Wensch-Dorendorf, M.; Titze, N.; Rodehutscord, M.; Rupp, C.; Susenbeth, A.; Greef, J.M.; Zeyner, A. Feed Clusters According to In Situ and In Vitro Ruminal Crude Protein Degradation. Animals 2023, 13, 224. [Google Scholar] [CrossRef]
- Abdelgadir, I.E.; Cochran, R.C.; Titgemeyer, E.C.; Vanzant, E.S. In Vitro Determination of Ruminal Protein Degradability of Alfalfa and Prairie Hay via a Commercial Protease in the Presence or Absence of Cellulase or Driselase. J. Anim. Sci. 1997, 75, 2215. [Google Scholar] [CrossRef]
- Assoumani, M.B.; Vedeau, F.; Jacquot, L.; Sniffen, C.J. Refinement of an Enzymatic Method for Estimating the Theoretical Degradability of Proteins in Feedstuffs for Ruminants. Anim. Feed Sci. Technol. 1992, 39, 357–368. [Google Scholar] [CrossRef]
- Pedersen, M.B.; Dalsgaard, S.; Arent, S.; Lorentsen, R.; Knudsen, K.E.B.; Yu, S.; Lærke, H.N. Xylanase and Protease Increase Solubilization of Non-Starch Polysaccharides and Nutrient Release of Corn- and Wheat Distillers Dried Grains with Solubles. Biochem. Eng. J. 2015, 98, 99–106. [Google Scholar] [CrossRef]
- Cone, J.W.; van Gelder, A.H.; Steg, A.; Van Vuuren, A.M. Prediction of in Situ Rumen Escape Protein from in Vitro Incubation with Protease from Streptomyces Griseus. J. Sci. Food Agric. 1996, 72, 120–126. [Google Scholar] [CrossRef]
- Guerra, N.P. Enzyme Kinetics Experiment with the Multienzyme Complex Viscozyme L and Two Substrates for the Accurate Determination of Michaelian Parameters. J. Chem. Educ. 2017, 94, 795–799. [Google Scholar] [CrossRef]
- Tománková, O.; Kopečný, J. Prediction of Feed Protein Degradation in the Rumen with Bromelain. Anim. Feed Sci. Technol. 1995, 53, 71–80. [Google Scholar] [CrossRef]
- Saleh, F.; Ohtsuka, A.; Tanaka, T.; Hayashi, K. Effect of Enzymes of Microbial Origin on in Vitro Digestibilities of Dry Matter and Crude Protein in Maize. J. Poult. Sci. 2003, 40, 274–281. [Google Scholar] [CrossRef]
- Saleh, F.; Ohtsuka, A.; Tanaka, T.; Hayashi, K. Carbohydrases Are Digested by Proteases Present in Enzyme Preparations During in Vitro Digestion. J. Poult. Sci. 2004, 41, 229–235. [Google Scholar] [CrossRef]
- Rosset, M.; Prudencio, S.H.; Beléia, A.D.P. Viscozyme L Action on Soy Slurry Affects Carbohydrates and Antioxidant Properties of Silken Tofu. Food Sci. Technol. Int. 2012, 18, 531–538. [Google Scholar] [CrossRef] [PubMed]
- Bravo Rodriguez, V.; Jurado Alameda, E.; Martinez Gallegos, J.F.; Reyes Requena, A.; Garcia Lopez, A.I.; Cabral, J.M.S.; Fernandes, P.; Da Fonseca, L.J.P. Modification of the Activity of an A-Amylase from Bacillus Licheniformis by Several Surfactants. Electron. J. Biotechnol. 2006, 9, 567–571. [Google Scholar] [CrossRef]
- Edmunds, B.; Südekum, K.-H.; Spiekers, H.; Schwarz, F.J. Estimating Ruminal Crude Protein Degradation of Forages Using in Situ and in Vitro Techniques. Anim. Feed Sci. Technol. 2012, 175, 95–105. [Google Scholar] [CrossRef]
- Simon, A.H.; Liebscher, S.; Aumüller, T.H.; Treblow, D.; Bordusa, F. Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases. Front. Microbiol. 2019, 10, 711. [Google Scholar] [CrossRef] [PubMed]
- Atherton, E.; Fox, H.; Harkiss, D.; Logan, C.J.; Sheppard, R.C.; Williams, B.J. A Mild Procedure for Solid Phase Peptide Synthesis: Use of Fluorenylmethoxycarbonylamino-Acids. J. Chem. Soc. Chem. Commun. 1978, 13, 537. [Google Scholar] [CrossRef]
- Merrifield, R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149–2154. [Google Scholar] [CrossRef]
- King, B.C.; Donnelly, M.K.; Bergstrom, G.C.; Walker, L.P.; Gibson, D.M. An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall-Degrading Enzyme Activity of Fungal Culture Extracts. Biotechnol. Bioeng. 2009, 102, 1033–1044. [Google Scholar] [CrossRef]
- Ghose, T.K. Measurement of Cellulase Activities. Pure Appl. Chem. 1987, 59, 257–268. [Google Scholar] [CrossRef]
- Bisswanger, H. Enzyme Assays. Perspect. Sci. 2014, 1, 41–55. [Google Scholar] [CrossRef]
- Zhang, S.; Vardhanabhuti, B. Intragastric Gelation of Whey Protein–Pectin Alters the Digestibility of Whey Protein during in Vitro Pepsin Digestion. Food Funct. 2014, 5, 102–110. [Google Scholar] [CrossRef] [PubMed]
- Jiang, L.; Yuan, C.; Huang, M. A General Strategy to Inhibit Serine Protease by Targeting Its Autolysis Loop. FASEB J. 2021, 35, e21259. [Google Scholar] [CrossRef] [PubMed]
IQFS 1 + | p-Value | IQFS 1 + | p-Value | IQFS 2 + | p-Value | IQFS 2 + | p-Value | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Time (min) | Buffer | Buffer + Protease | Time × Variant | Pectin | Pectin + Protease | Time × Variant | Buffer | Buffer + Protease | Time × Variant | Pectin | Pectin + Protease | Time × Variant |
0 | 12.2 b | 14.3 a | <0.0001 | 12.6 a | 13.3 a | 0.4883 | 12.9 b | 16.7 a | 0.0005 | 14.7 a | 13.6 a | 0.3769 |
1 | 12.2 b | 18.4 a | <0.0001 | 12.6 a | 13.8 a | 0.2105 | 12.9 b | 21.1 a | <0.0001 | 14.8 a | 14.0 a | 0.5406 |
2 | 12.2 b | 22.4 a | <0.0001 | 12.8 a | 14.6 a | 0.0689 | 12.8 b | 25.8 a | <0.0001 | 15.1 a | 15.1 a | 0.9771 |
3 | 12.2 b | 26.0 a | <0.0001 | 12.7 b | 15.5 a | 0.0040 | 12.8 b | 30.0 a | <0.0001 | 14.9 a | 15.6 a | 0.5933 |
4 | 12.1 b | 29.5 a | <0.0001 | 12.5 b | 16.2 a | 0.0001 | 12.8 b | 33.4 a | <0.0001 | 14.7 a | 16.3 a | 0.2260 |
5 | 12.1 b | 32.6 a | <0.0001 | 12.7 b | 17.2 a | <0.0001 | 12.7 b | 37.6 a | <0.0001 | 14.7 a | 17.3 a | 0.0640 |
6 | 12.1 b | 35.8 a | <0.0001 | 12.8 b | 17.9 a | <0.0001 | 12.8 b | 41.0 a | <0.0001 | 14.8 b | 18.4 a | 0.0096 |
7 | 12.2 b | 38.1 a | <0.0001 | 12.6 b | 19.2 a | <0.0001 | 12.8 b | 43.4 a | <0.0001 | 14.6 b | 19.7 a | 0.0003 |
8 | 12.2 b | 40.9 a | <0.0001 | 12.5 b | 20.4 a | <0.0001 | 12.8 b | 45.5 a | <0.0001 | 14.6 b | 21.5 a | <0.0001 |
9 | 12.3 b | 42.2 a | <0.0001 | 12.6 b | 21.6 a | <0.0001 | 12.8 b | 47.8 a | <0.0001 | 14.7 b | 23.0 a | <0.0001 |
10 | 12.2 b | 44.7 a | <0.0001 | 12.5 b | 22.8 a | <0.0001 | 12.7 b | 49.9 a | <0.0001 | 14.6 b | 25.1 a | <0.0001 |
SD | 0.1–0.2 | 0.0–1.2 | - | 0.2–0.5 | 0.5–2.3 | - | 0.8–1.2 | 0.5–1.5 | - | 0.1–0.3 | 1.1–2.8 | - |
Reducing Sugar Concentration | p-Value | |||
---|---|---|---|---|
Time (h) | Pectin | Pectin + Viscozym L | Pectin + Viscozym L + Protease | Time × Variant |
0 | 0.3 | 0 a | 0 a | 10.000 |
1 | 0 | 2.6 a | 2.5 a | 0.9889 |
2 | 0 | 4.6 a | 4.1 a | 0.1636 |
3 | 0 | 4.7 b | 5.4 a | 0.0304 |
5 | 0 | 5.9 a | 5.8 a | 0.6590 |
Range of SD | 0–0.1 | 0–0.5 | 0–0.7 | - |
Reducing Sugar Concentration | p-Value | |||
---|---|---|---|---|
Time (h) | Starch | Starch + α-Amylase | Starch + α-Amylase + Protease | Time × Variant |
0 | 0.1 | 0 a | 0 a | 10.000 |
1 | 0.1 | 4.2 a | 4.0 a | 0.6507 |
2 | 0.1 | 6.2 a | 5.9 a | 0.4366 |
3 | 0.1 | 7.0 a | 7.1 a | 0.9124 |
5 | 0.1 | 9.2 a | 8.9 a | 0.5111 |
Range of SD | 0 | 0–0.6 | 0–1.3 | - |
Reducing Sugar Concentration | p-Value | |||
---|---|---|---|---|
Time (h) | Starch | Starch + Viscozym L | Starch + Viscozym L + Protease | Time × Variant |
0 | 0.2 | 0 a | 0 a | 10.000 |
1 | 0.1 | 0.9 a | 0.3 a | 0.3150 |
2 | 0.2 | 1.2 a | 0.9 a | 0.5643 |
3 | 0.1 | 1.3 a | 1.1 a | 0.6843 |
5 | 0.1 | 2.7 a | 1.7 a | 0.0680 |
Range of SD | 0 | 0–1.1 | 0–0.9 | - |
Reducing Sugar Concentration | p-Value | |||
---|---|---|---|---|
Time (h) | Cellulose | Cellulose + Viscozym L | Cellulose + Viscozym L + Protease | Time × Variant |
0 | 0 | 0.1 a | 0.6 a | 0.3379 |
1 | 0.1 | 0.8 a | 0.5 a | 0.5868 |
2 | 0 | 0.9 a | 0.8 a | 0.9552 |
3 | 0.1 | 1.3 a | 1.2 a | 0.7714 |
5 | 0 | 1.7 a | 1.6 a | 0.9009 |
Range of SD | 0–0.1 | 0.1–1.2 | 0.4–1.1 | - |
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Okon, P.; Liebscher, S.; Simon, A.H.; Wensch-Dorendorf, M.; Bachmann, M.; Bordusa, F.; Zeyner, A. The Impact of Streptomyces griseus Protease Reserved for Protein Evaluation of Ruminant Feed on Carbohydrase Activity during Co-Incubation. Animals 2024, 14, 1931. https://doi.org/10.3390/ani14131931
Okon P, Liebscher S, Simon AH, Wensch-Dorendorf M, Bachmann M, Bordusa F, Zeyner A. The Impact of Streptomyces griseus Protease Reserved for Protein Evaluation of Ruminant Feed on Carbohydrase Activity during Co-Incubation. Animals. 2024; 14(13):1931. https://doi.org/10.3390/ani14131931
Chicago/Turabian StyleOkon, Paul, Sandra Liebscher, Andreas Hans Simon, Monika Wensch-Dorendorf, Martin Bachmann, Frank Bordusa, and Annette Zeyner. 2024. "The Impact of Streptomyces griseus Protease Reserved for Protein Evaluation of Ruminant Feed on Carbohydrase Activity during Co-Incubation" Animals 14, no. 13: 1931. https://doi.org/10.3390/ani14131931
APA StyleOkon, P., Liebscher, S., Simon, A. H., Wensch-Dorendorf, M., Bachmann, M., Bordusa, F., & Zeyner, A. (2024). The Impact of Streptomyces griseus Protease Reserved for Protein Evaluation of Ruminant Feed on Carbohydrase Activity during Co-Incubation. Animals, 14(13), 1931. https://doi.org/10.3390/ani14131931