Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

نویسندگان

  • Bradley J Stevenson
  • Christopher C Waller
  • Paul Ma
  • Kunkun Li
  • Adam T Cawley
  • David L Ollis
  • Malcolm D McLeod
چکیده

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Purification and characterization of the arylsulfatase synthesized by Pseudomonas aeruginosa PA0 during growth in sulfate-free medium and cloning of the arylsulfatase gene

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PA01 and purified 2700fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDSPAGE to be 57 kDa, and the enzyme was presumed to b...

متن کامل

EXTRACELLULAR LIPA SE ACTIVITY CHARACTERIZATION OF SOME PSEUDOMONAS AERUGINOSA STRA INS ISOLATED FROM HUMAN INFECTIONS

Pseudomonas aeruginosa EF2, ATCC 9027 and ATCC 19660 were grown in a continuous culture under Tween 80 (polyoxyethylene sorbitan monooleate) limitation and optimum conditions (pH 6.5, 37°C at dilution rate of 0.05/h). Culture supernatants were carefully removed and stored at -20°C. To purify the lipases, the culture supernatant was reduced in volume to approximately 10 mL by an ultrafiltra...

متن کامل

One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm

Objective(s): Pseudomonas aeruginosais a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Algin...

متن کامل

Purification and Characterization of Alginate Lyase from Mucoid Pseudomonas aeruginosa Strain 214

Pseudomonas aeruginosa is an opportunistic pathogen that causes a variety of infections in compromised patients. The ability of Pseudomonas aeruginosa to produce chronic infection is based in part on its ability to biosynthesis of biofilm, and alginate is the major polysaccharide in the synthesized biofilm. So alginate degradation is very essential in the dispersion of Pseudomonas aeruginosa bi...

متن کامل

Optimization of Lipase Immobilization

Pseudomonas aeruginosa BBRC-10036 was used for lipase production. The organism secreted the enzyme extracellulary. In order to purify the enzyme, precipitation was done first, and then this lipase has been purified by Ion exchange Chromatography leading to 2.3-fold purification and 11.47% recovery. Lipase from P.aeruginosa was entrapped into Ca-alginate gel beads and effect of independent varia...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Drug testing and analysis

دوره 7 10  شماره 

صفحات  -

تاریخ انتشار 2015