Search

Proteolytic enzymes are used for proteolysis and peptide synthesis which can be run in various conditions including low pH value and the presence of ethanol. The most common cysteine protease applied in acidic-alcoholic conditions is well-characterized papain. Ficin, which is closely related to papain in terms of proteolytic activity and substrate specificity, could potentially be applied in the alcoholic beverage industry and peptide synthesis. The aim of this study was to compare papain and ficin stability in process conditions. Comparative stability study showed that ficin as a mixture of different isoforms has a broader range of stability in respect of pH and cold storage stability, in comparison to papain. It retains about 70% of initial activity after 3-week cold storage at low pH and in the presence of ethanol. Unlike ficin, papain loses about 70% of initial activity in the same incubation period as it is more prone to non-native aggregation that was confirmed by FTIR analysis. The presence of multiple isoforms of ficin stabilizes the protease against cold denaturation and aggregation, making it more suitable for biotechnological and laboratory usage than single papain isoform. It is more cold-stable in alcoholic-acidic and acidic conditions suggesting possible replacement of papain with even lower enzyme concentration. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3933]

Denaturacija globularnih proteina na niskim temperaturama predstavlja univerzalni fenomen. Narušavanje nativne strukture proteina usled izlaganja niskim temperaturama dešava se primarno kao posledica kolapsa hidrofobnog efekta, entropijskog faktora koji u najvećoj meri doprinosi stabilizaciji nativne strukture. Sekundarno, denaturacija proteina na niskim temperaturama posledica je slabljenja hidrofobnih interakcija u unutrašnjosti nativnog proteina. Ispitivanje stabilnosti proteina na niskim temperaturama ima, pre svega, fundamentalni značaj koji se ogleda u činjenici da bi detaljno razumevanje mehanizma denaturacije proteina na niskim temperaturama moglo značajno da doprinese razjašnjavanju jednog od najvažnijih problema savremene biohemije, problema uvijanja proteina. Ispitivanje stabilnosti proteina na niskim temperaturama započeto je pre oko dve decenije. Međutim, direktnu denaturaciju proteina, koja je posledica niske temperature per se, teško je proučavati rutinski primenjivanim metodama, jer većina proteina ima tačke denaturacije ispod temperature mržnjenja vode. Razvojem savremenih FT-IR instrumenata, kao i primenom istih u određivanju promena sekundarnih struktura proteina, došlo je do povećanog interesovanja za proučavanjem denaturacije proteina izazvane niskim temperaturama. Praktični značaj proučavanja ove problematike ogleda se u tome što razumevanje mehanizama denaturacije može pomoći u pronalaženju optimalnih uslova za skladištenje proteina pri kojima će se produžiti njihov vek trajanja. Poznavanje stabilnosti enzima naročito je važno u slučaju enzima koji se koriste u biotehnologiji, medicini ili nauci, kao što je slučaj sa sve četiri model proteaze koje su predmet istraživanja u okviru ove doktorske disertacije. Najvažniji cilj koji je postavljen u ovoj studiji jeste pronalaženje objašnjenja za gubitak aktivnosti izazvan niskom temperaturom ispitivanih serin- i cistein-proteaza na nivou detaljnih strukturnih promena proteina, odnosno, pokazati da su, zapravo, denaturacija proteaza i strukturni rearanţmani koji prate denaturaciju odgovorni za gubitak aktivnosti u mnogo većoj meri nego autoproteoliza. Dodatni cilj bilo je optimizovanje uslova za skladištenje komercijalno vaţnih proteaza na niskim temperaturama. Praćenje stabilnosti proteina na niskim temperaturama nailazi na metodološka ograničenja kada je reč o primeni uobičajeno korišćenih metoda za ispitivanje stabilnosti proteina, kao što su spektroskopske metode i diferencijalna skenirajuća kalorimetrija. Stoga je u ovoj studiji pribegnuto pristupu izlaganja proteaza uzastopnim ciklusima zamrzavanja/odmrzavanja u cilju praćenja uticaja temperatura ispod nule na strukturu proteaza. Rezultati praćenja promena primarne strukture model proteaza koji su dobijeni kao deo ove disertacije, ukazuju da autoproteoliza ne može biti odgovorna za visoke procente gubitka aktivnosti, (čak do 75% u slučaju papaina, preko 40% u slučaju ficina i oko 60% u slučaju tripsina u kiselim uslovima) nakon šest ciklusa zamrzavanja/odmrzavanja. Praćenje strukturnih perturbacija na nivou sekundarne i tercijarne strukture pokazalo je da nakon šest do sedam uzastopnih ciklusa zamrzavanja/odmrzavanja proteaze gube elemente nativne sekundarne strukture (α- heliks i neuređene strukture) u korist β-pločica (intramolekulskih u slučaju tripsina u kiselim uslovima). Naročito je izražen porast sadržaja intermolekulskih β-pločica (u slučaju papaina i ficina) koje predstavljaju strukturne elemente neophodne za agregiranje. Agregiranje je potvrđeno gel-filtracijom. Opisani trendovi promena sekundarnih struktura detektovani su u literaturi i za druge proteine denaturisane niskim temperaturama, odnosno niskom pH vrednošću, indicirajući da je inaktiviranje papaina, ficina i tripsina (u kiselim uslovima) na niskoj temperaturi posledica denaturacije. U slučaju tripsina za sekvenciranje, proteaze od najvećeg komercijalnog značaja u ovoj studiji, predložen je alternativni način skladištenja na niskoj temperaturi u rastvornom obliku kojim se izbegava denaturacija i ograničava autoproteoliza. Blago alkalna pH vrednost (u blizini optimalne vrednosti za aktivnost tripsina) uz dodatak krioprotektivnih agenasa (glicerola i lizina) za koje je poznato da stabilizuju proteine (mehanizmom preferencijalne potisnutosti sa površine proteina favorizujući nativnu konformaciju), dovela je do efikasnog očuvanja nativne strukture tripsina. Takođe, inhibicijom autoproteolitičke aktivnosti u prisustvu lizina koji okupira vezujuće mesto tripsina, ograničena je i autoproteoliza. Tripsin skladišten na niskoj temperaturi na pH vrednosti od 8,0 uz dodatak glicerola ili lizina, pokazao je efikasnost identičnu netretiranom nativnom tripsinu u metodi identifikovanja BSA peptidnim mapiranjem, što sugeriše da skladištenje tripsina u blago alkalnim uslovima uz dodatak krioprotektanata može da produži njegov vek trajanja. Cold denaturation of globular proteins represents a universal phenomenon. Disruption of native structure at low temperatures primarily happens as a consequence of the collapse of the hydrophobic effect, an entropy parameter which represents a main driving force for protein folding. Secondarily, protein denaturation at low temperatures is a consequence of the weakening of hydrophobic interactions in the interior of a protein's three dimensional structure. Investigation of the cold denaturation of proteins has fundamental importance, because detailed understanding of the mechanism of cold denaturation could contribute to a great extent toward elucidationg one of the most challenging problems of contemporary structural biochemistry: the protein folding problem. Investigation of the cold stability of proteins began two decades ago. Direct cold denaturation, which is a consequence of low temperature per se, is difficult to investigate using routine methods since the majority of proteins have cold denaturation points well below 0 °C. However, the development of modern FT-IR instruments and their application in secondary structure determination has lead to increased interest in the investigation of the cold stability of proteins. The practical importance of this topic reflects the fact that understanding structural rearrangements induced by cold denaturation could help define optimal conditions for protein storage in order to prolong their shelf lives. The cold stability of proteins is especially important for enzymes used in biotechnology, medicine or research: which is the case for all four model proteases investigated in this dissertation. The overall goal of this study was to explain the dramatic loss of serine and cysteine proteases activity induced by low temperature, by showing that cold denaturation and subsequent massive structural rearrangements are responsible for this loss, rather than autoproteolysis. In addition, optimal cold storage conditions were defined for commercially important proteases. In this work, repeated freeze-thaw cycles were used to analyse the influence of sub-zero temperatures on protease structures, due to methodological limitations in monitoring protein stability at sub-zero temperatures by commonly used techniques such as spectroscopy and differential scanning calorimetry. Results from monitoring the primary structure stability of model proteases obtained as a part of this dissertation suggest that autoproteolysis cannot be the cause for the dramatic activity losses (as large as 75% in the case of papain, above 40% in the case of ficin and around 60% in the case of trypsin in acidic conditions) observed after six freeze-thaw cycles. Secondary/tertiary structure perturbations after six-to-seven freeze-thaw cycles suggest that these model proteases lose part of their native secondary structure elements (mainly α-helices and random coils) in favor of a β-sheet conformation (intramolecular in the case of trypsin in acidic conditions). An especially large increase was detected for intermolecular β- sheets (in the case of papain and ficin) which represents a structural element necessary for aggregation. Aggregation of cold denatured cysteine proteases was shown by sizeexclusion chromatography. Similar trends in secondary structure changes were detected in the literature for other proteins denatured by low temperature and/or low pH values, indicating that inactivation of papain, ficin and trypsin (in acidic conditions) was a consequence of cold denaturation. Sequencing grade trypsin, being the most important commercial protease of this study, was chosen for optimization of cold storage conditions. An alternative protocol for cold storage of trypsin in solution is proposed which circumvents cold denaturation and limits autoproteolysis. Slightly alkaline conditions (close to the optimum pH value of trypsin, pH around 8) with the addition of cryoprotectants (glycerol or lysine which are known to stabilise proteins in solution by preferential exclusion from the protein surface favoring the native state) led to complete preservation of native structure. In the case of lysine as a cryoprotectant, autoproteolysis was inhibited as well. After seven cycles of cold storage at pH around 8 with the addition of cryoprotectants, trypsin was as efficient as untreated trypsin in trypsin mass fingerprinting of BSA, suggesting that proposed conditions could prolong its shelf life.

Trypsin is a serine protease with widespread applications, including protein sequencing and typsin 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 beta-sheet content (band maximum detected at a frequency of 1634 cm 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 ayoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery ( gt 90%) and modest autolysis ( lt 10%). High activity recovery ( gt 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. (C) 2015 Elsevier B.V. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3586]

Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 degrees C to 60 degrees C with Delta G degrees(321) of 13.9 +/- 0.3 kJ/mol and T-m value of 84 +/- 1 degrees C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular beta-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry. This is the peer-reviewed version of the following article: Rašković, B.; Popović, M.; Ostojić, S.; Ancrossed D Signelković, B.; Tešević, V.; Polović, N. Fourier Transform Infrared Spectroscopy Provides an Evidence of Papain Denaturation and Aggregation during Cold Storage. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2015, 150, 238–246. [https://doi.org/10.1016/j.saa.2015.05.061]

In winter flounder, the levels of type I antifreeze protein (AFP) and its mRNA vary seasonally by as much as 1000-fold. Elevated levels in the fall are prompted by the loss of long day-lengths, while higher spring temperatures correlate with AFP clearance. We have investigated the role of temperature on AFP accumulation using transgenic Drosophila melanogaster by expressing multiple AFP genes under control of the heat-inducible hsp70 promoter. AFP and AFP mRNA persisted far longer in flies reared at 10 degrees C compared to 22 degrees C. This difference appears to be mediated by cold-specific mRNA stability since no such temperature effect was observed with either an endogenous heat-inducible mRNA or a constitutively expressed mRNA.

The amount of axonally transported proteins in the nervus splanchnicus of cod (Gadus morhua) was found to be temperature-dependent in vitro, with an optimum at 8°C. The transport was markedly reduced at 2°C, probably caused by decreased protein synthesis rather than disassembly of microtubules. Microtubules were isolated from cod brain by cycles of assembly-disassembly. These microtubules were cold-labile, had a low amount of microtubule-associated proteins and a high critical concentration for assembly. The possibilities exist that a cold-stabilizing factor or cold-stable microtubules are lost during the preparation, or that cold-stable microtubules are components of the peripheral axons only.

Cold-stable and cold-labile microtubules were prepared by two cycles of assembly and disassembly and two periods of exposure to cold. The cold-labile preparations were shown to contain a higher proportion of a high molecular mass microtubule-associated protein (MAP 2) than cold-stable preparations. The cold-stable preparations showed a much higher alkaline phosphatase activity. Stimulation of microtubule assembly by zinc led to increases in both cold stability and alkaline phosphatase activity.