Optimizacija izolacije rekombinantne serinske proteinaze pernizin iz bakterije Streptomyces rimosus

Špehar, Ivana (author), Poklar Ulrih, Nataša (mentor), Šnajder, Marko (comentor)

Cilj magistrskega dela je bil, da izboljšamo postopek izolacije rekombinantnega pernizina s pravilno izbiro ter optimizacijo tehnik izolacije in s tem izboljšamo izkoristek produkta v sistemu za izražanje proteinov Streptomyces rimosus kot tudi, da opišemo fizikalne lastnosti in encimsko aktivnost pernizina glede na različne parametre, kot so temperatura in pH. Za izražanje rekombinantnega pernizina smo uporabili bakterijo Streptomyces rimosus M4081, katero smo transformirali s plazmidom pVF tcp830 srT pernisine CO HT. Pernizin smo očistili najprej z afinitetno kromatografijo NiNTA, nadalje pa smo še izvedli korake velikostno izključitvene kromatografije, dialize in koncentriranja vzorca pernizina in s tem dobili še boljšo čistost pernizina, kar smo pokazali z NaDS-PAGE ter cimografijo. Njegove fizikalne lastnosti smo analizirali s CD- spektropolarimetrijo in fluorescenčno emisijsko spektrometrijo ter smo pokazali, da rekombinantni pernizin ohrani strukturno konformacijo pri razponu temperatur od 25 do 60 °C ter v območju pH od 3,0 do 12,0. Encimsko aktivnost rekombinantnega pernizina smo preverili z azokazeinskim testom in smo pokazali, da je rekombinantni pernizin termostabilen, saj ohrani encimsko aktivnost večjo kot 90 % pri temperaturi 80 °C v časovnem obdobju 240 minut. Preostalo encimsko aktivnost, ki je večja kot 80 % ima v širokem razponu pH od 5,3 do 10,1 pri temperaturi 100 °C, z maksimumom pri vrednosti pH 6,95 ter temperaturi 100 °C. V zaključku smo dokazali, da vrednost encimske aktivnosti rekombinantnega pernizina, pri določenih stopnjah izolacije ustreza ravni čistosti pernizina, pridobljenega s posameznimi stopnjami izolacije.

• Language: Slovenian
• Keywords: optimizacija izolacije, Streptomyces rimosus, rekombinantni proteini, serinska proteinaza, pernizin
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [I. Špehar]
• Year: 2018
• PID: 20.500.12556/RUL-105889
• UDC: 602.3:579.873.7+604.6:577.15
• COBISS.SI-ID: 5012088
• Publication date in RUL: 22.12.2018

ŠPEHAR, Ivana, 2018, Optimizacija izolacije rekombinantne serinske proteinaze pernizin iz bakterije Streptomyces rimosus [online]. Master’s thesis. I. Špehar. [Accessed 11 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=105889

Secondary language

• Language: English
• Title: Optimization of isolation of recombinant serine proteinase pernisine from Streptomyces rimosus

Abstract

The aim of the master's thesis was to improve the efficiency of the isolation of recombinant pernisine from the Streptomyces rimosus expression system by correctly selecting and optimizing the procedures for the isolation and describing the physical properties and enzymatic activity of recombinant pernisine with regard to various parameters such as temperature and pH. Streptomyces rimosus M4081 was transformed to express recombinant pernisine, transformed with plasmid pVF tcp830 srT srT pernisine CO HT. The first step in the isolation of pernisine was performance of Ni2+affinity chromatography, followed by the steps of size exclusion chromatography, dialysis and concentration of the pernisine sample, thereby obtaining even better purity of pernisine, as demonstrated by NaDS-PAGE and zymography. Physical properties were analyzed by CD-spectrometry and fluorescence emission spectrometry, and it has been demonstrated that recombinant pernisine maintains structural conformation well at a temperature range of 25 to 60 °C and in a pH range of 3.0 to 12.0. The enzyme activity of recombinant pernisine was tested with an azocasein test and it has been demonstrated that recombinant pernisne is thermostable because it retains the residual enzymatic activity greater than 90 % at a temperature of 80 °C over a period of 240 minutes. Recombinant pernizine maintains the ramaining enzymatic activity that is greater than 80%, in a wide range of pH from 5.3 to 10.1 at a temperature of 100 °C with the maximum at pH 6.95 and at a temperature of 100 °C. In conclusion, we have shown that the value of the enzymatic activity of recombinant pernisine, at certain levels of isolation, corresponds to the level of purity of the pernisine obtained by the individual levels of isolation.

• Keywords: optimization of isolation, Streptomyces rimosus, recombinant proteins, serine proteinase, pernisine