Purification, characterization and comparison of Penicillium purpurogenum β‐glucuronidases expressed in Escherichia coli and Pichia pastoris. Issue 10 (29th April 2013)

• Record Type: Journal Article
• Authors: Zou, Shuping; Guo, Shuyuan; Kaleem, Imdad; Li, Chun
• Is Part Of: Journal of chemical technology & biotechnology. Volume 88:Issue 10(2013:Oct.)
• Page Start: 1913
• Page End: 1919
• Publication Date: 2013-04-29
• Subjects: Biotechnology -- Periodicals; Chemistry, Technical -- Periodicals; Chemical engineering -- Periodicals; Industries -- Environmental aspects -- Periodicals; 660
• DOI: 10.1002/jctb.4050
• Languages: English
• ISSNs: 0268-2575

Abstract

<abstract abstract-type="main"> <title>Abstract</title> <sec id="jctb4050-sec-0001" sec-type="section"> <title>Background</title> <p> <bold>To investigate the effects of post‐translational modifications in different recombinant expression systems on the catalytic properties of recombinant β‐glucuronidase. The β‐glucuronidase (GUS) gene from <italic>Penicillium purpurogenum</italic> Li‐3 was cloned and successfully expressed in <italic>Escherichia coli</italic> BL21 and <italic>Pichia pastoris</italic> G115</bold>.</p> </sec> <sec id="jctb4050-sec-0002" sec-type="section"> <title>Results</title> <p> <bold>The recombinant <italic>E. coli</italic> produced a 15‐fold increased level of β‐glucuronidase while the recombinant <italic>P. pastoris</italic> strain produced a 6.9‐fold increased level of β‐glucuronidase compared with their parent strains. The β‐glucuronidases from recombinant <italic>E. coli</italic> (PGUS‐E) and <italic>P. pastors</italic> (PGUS‐P) were purified to 35.9‐ and 47.4‐fold, respectively, through affinity, ion exchange and gel filtration chromatography. PGUS‐E from <italic>E. coli</italic> was a non‐glycosylated protein with an apparent molecular mass of 72.43 kDa, while PGUS‐P from <italic>P. pastors</italic> was appropriately glycosylated with a molecular mass of 78.83 kDa measured by MALDI/TOF‐MS. Although both recombinant β‐glucuronidases exhibited similar pH optima, the glycosylated PGUS‐P showed a significantly higher thermal stability and less sensitivity to metal ions compared with the non‐glycosylated PGUS‐E. The glycosylated PGUS‐P also displayed lower <italic>K</italic><sub>m</sub> values, and higher <italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>m</sub> ratios than the non‐glycosylated enzyme towards glycyrrhizin</bold>.</p> </sec> <sec id="jctb4050-sec-0003" sec-type="section"> <title>Conclusion</title> <p> <bold>These results revealed the key role of post‐translational modifications in the <italic>P. pastors</italic> expression system on the catalytic properties of β‐glucuronidase and its potential stability over the prokaryotic expression system which could be applied as an important tool for the functional enhancement of industrial enzymes. © 2013 Society of Chemical Industry</bold> </p> </sec> </abstract>