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Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life

Authorized Users Only
2016
Authors
Rašković, Brankica
Vatić, Saša
Anđelković, Boban
Blagojević, Vladimir A.
Polović, Natalija
Article (Published version)
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Abstract
Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze–thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in β-sheet content (band maximum detected at a frequency of 1634 cm−1 in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, t...rypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life.

Keywords:
Enzyme activity / Cold stability / Protein recovery / Protein denaturation / Proteolysis
Source:
Biochemical Engineering Journal, 2016, 105, A, 168-176
Publisher:
  • Elsevier
Projects:
  • Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance (RS-172049)
  • Directed synthesis, structure and properties of multifunctional materials (RS-172057)

DOI: 10.1016/j.bej.2015.09.018

ISSN: 1369-703X

WoS: 000367776300019

Scopus: 2-s2.0-84942792521
[ Google Scholar ]
3
2
URI
http://dais.sanu.ac.rs/123456789/16003
Collections
  • ITN SANU - Opšta kolekcija / ITS SASA - General collection
Institution
Институт техничких наука САНУ / Institute of Technical Sciences of SASA
TY  - JOUR
AU  - Rašković, Brankica
AU  - Vatić, Saša
AU  - Anđelković, Boban
AU  - Blagojević, Vladimir A.
AU  - Polović, Natalija
PY  - 2016
UR  - http://dais.sanu.ac.rs/123456789/16003
AB  - Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze–thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in β-sheet content (band maximum detected at a frequency of 1634 cm−1 in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life.
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life
SP  - 168
EP  - 176
VL  - 105
IS  - A
DO  - 10.1016/j.bej.2015.09.018
ER  - 
@article{
author = "Rašković, Brankica and Vatić, Saša and Anđelković, Boban and Blagojević, Vladimir A. and Polović, Natalija",
year = "2016",
url = "http://dais.sanu.ac.rs/123456789/16003",
abstract = "Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze–thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in β-sheet content (band maximum detected at a frequency of 1634 cm−1 in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life.",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life",
pages = "168-176",
volume = "105",
number = "A",
doi = "10.1016/j.bej.2015.09.018"
}
Rašković B, Vatić S, Anđelković B, Blagojević VA, Polović N. Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life. Biochemical Engineering Journal. 2016;105(A):168-176
Rašković, B., Vatić, S., Anđelković, B., Blagojević, V. A.,& Polović, N. (2016). Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life.
Biochemical Engineering JournalElsevier., 105(A), 168-176. 
https://doi.org/10.1016/j.bej.2015.09.018
Rašković Brankica, Vatić Saša, Anđelković Boban, Blagojević Vladimir A., Polović Natalija, "Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life" 105, no. A (2016):168-176,
https://doi.org/10.1016/j.bej.2015.09.018 .

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