Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

Santos, Alexandre Martins Costa; Oliveira, Jamil S.; Bittar, Eustáquio Resende; Silva, Anderson Lourenço da; Guia, Marcos Luiz dos Mares; Bemquerer, Marcelo P.; Santoro, Marcelo M.

doi:10.1590/s1516-89132008000400009

Cited by 10 publications

(7 citation statements)

References 14 publications

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“…Elution of several peaks in AEC with the same molecular weight in SDS‐PAGE is probably due to the presence of enzyme isoforms. This type of chromatography has been reported as a method that enables to separate proteases isoforms . Additionally, peaks P4 and P5 were identified as other two different enzymes named as EII and EIII and with molecular weight of 40 and 55 kDa, respectively.…”

Section: Resultssupporting

confidence: 92%

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles

Flores-Fernández

Cárdenas-Fernández

Dobrijevic

et al. 2018

Biotechnology Progress

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Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified. EI and EIII were strongly inhibited by EDTA and Pefabloc being classified as serine‐metalloproteases, while EII was completely inhibited only by Pefabloc being classified as a serine protease. In addition, EI and EII exhibited highest enzymatic activity at pH 8, while EIII at pH 11 maintaining almost 100% of it at pH 12. All the enzymes demonstrated optimum activity at 60°C. Enzymatic activity of EI was strongly stimulated in presence of Mn2+ (6.9‐fold), EII was stimulated by Mn2+ (3.7‐fold), while EIII was slightly stimulated by Zn2+, Ca2+, and Mg2+. DDGS protein hydrolysis using purified Pseudomonas aeruginosa M211 proteases demonstrated that, based on glycine released, EIII presented the highest proteolytic activity toward DDGS. This enzyme enabled the release 63% of the total glycine content in wheat DDGS protein, 2.2‐fold higher that when using the commercial Pronase®. Overall, our results indicate that this novel extremopreoteases have a great potential to be applied in DDGS hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2728, 2019

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Section: Resultssupporting

confidence: 92%

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles

Flores-Fernández

Cárdenas-Fernández

Dobrijevic

et al. 2018

Biotechnology Progress

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“…All steps of the purification of trypsin isoforms were conducted according to the method of Santos et al [8]. After protein isolation, only the fractions with purity close to 100% of ␣-trypsin were used.…”

Section: Protein Purification and Characterizationmentioning

confidence: 99%

“…For this proposal, once for month (during 12 months) aliquots of ␣-trypsin were re-chromatographed at a similar system, but at a smaller scale than that previously described [8]. Resultant products were analyzed by UV absorption (280 nm) using the molar extinction coefficient value of 40,000 M −1 cm −1 and identified by mass spectrometry (Q-ToF, Micromass).…”

Section: Determination Of Transformation Percentage Of α-Trypsin Intomentioning

confidence: 99%

“…A second isoform of ␤-trypsin, which is present in smaller amount (in commercial preparations) is originated from hydrolysis of ␣-trypsin at the peptide bond between Lys-176 and Asn-177 producing -trypsin [5] that has three polypeptide chains linked by disulfide bond. Both ␤-and ␣-trypsin show significant amidasic activity (␤-trypsin is about 50% more active than ␣-trypsin under the conditions of hydrolytic assay of N ␣ -benzoyl-dl-arginine-4-nitroanilide [8]). However the -trypsin isoform presents some differences such as a higher affinity for positively charged ligands like N ␣ -benzoyl-l-arginine ethyl ester and benzamidine, and its poor amidasic activity [5].…”

Section: Introductionmentioning

confidence: 99%

“…However the -trypsin isoform presents some differences such as a higher affinity for positively charged ligands like N ␣ -benzoyl-l-arginine ethyl ester and benzamidine, and its poor amidasic activity [5]. The isoform ␣-trypsin can now be purified in large quantities [8] and it undergoes reversible thermally-induced or denaturant-induced transitions in acidic media. The aim of our work was to elucidate if there is thermodynamic significant differences between ␣-and ␤-trypsin isoforms at pH 3.0 [9] using direct measurements by differential scanning calorimetry (DSC).…”

Section: Introductionmentioning

confidence: 99%

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Physical-chemical characterization and stability study of α-trypsin at pH 3.0 by differential scanning calorimetry

Santos

Santana

Gomide

et al. 2008

International Journal of Biological Macromolecules

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Residual protein analysis by SDS–PAGE in clinically manufactured BM‐MSC products

Kilic,

Karabudak,

Cosar

et al. 2024

Electrophoresis

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Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue‐ and cell‐based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow‐derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) method. Despite being a semiquantitative method, SDS–PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS–PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS–PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost‐effective method for residual substance analyses in clinically manufactured cellular therapies.

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Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

Abstract: The purpose of this work was to improve the separation and yield of pure β-and α-trypsin isoforms by ion-

Cited by 10 publications

References 14 publications

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles

Physical-chemical characterization and stability study of α-trypsin at pH 3.0 by differential scanning calorimetry

Residual protein analysis by SDS–PAGE in clinically manufactured BM‐MSC products

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