Expression and Purification of Escherichia coli β-Glucuronidase

Aich, Sanjukta; Delbaere, L. T. J.; Chen, Ridong

doi:10.1006/prep.2001.1401

Cited by 10 publications

(10 citation statements)

References 15 publications

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“…7 shows the temperature profile for GUS catalysed hydrolysis of 4-MUG, 6-CMUG and 3-CUG. 19,43,44,[47][48][49] This behaviour is consistent with a simple diprotic system were the increase and decrease in activity on both sides of the optimum pH represents titration curves of active site residues. 42 Previous studies on wild type GUS using 4-nitrophenyl-β-D-glucuronide (4-NPG) and 4-MUG 43 as substrate 44,45 and recombinant GUS using 3-CUG as substrate 19,40 report similar temperature profiles with a maximum activity between 40-50°C.…”

Section: Temperature and Ph Optimisationsupporting

confidence: 75%

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Briciu-Burghina

Heery

Regan

2015

Analyst

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E. coli β-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl-umbelliferyl-β-D-glucuronide (6-CMUG) were determined experimentally (Km = 0.11 mM, Kcat = 74 s(-1), Kcat/Km = 6.93 × 10(5) s(-1) M(-1)) and compared with the ones found for 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) (Km = 0.07 mM, Kcat = 92 s(-1), Kcat/Km = 1.29 × 10(6) s(-1) M(-1)) and 3-carboxy-umbelliferyl-β-D-glucuronide (3-CUG) (Km = 0.48 mM, Kcat = 35 s(-1), Kcat/Km = 7.40 × 10(4) s(-1) M(-1)). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.

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Section: Temperature and Ph Optimisationsupporting

confidence: 75%

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Briciu-Burghina

Heery

Regan

2015

Analyst

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“…

β

‐Glucuronidase activity was measured at 25 °C using PNPG substrate . Para‐nitrophenol (PNP) product was measured at 405 nm in a Synergy H4 plate reader (BioTek Instruments, Winooski, VT, USA) with 1 unit of activity corresponding to production of 1 pmol PNP per minute. Activity in clarified conditioned medium was measured by diluting 10‐fold into 20 mM sodium phosphate, 1 mM PNPG, 10 mM

β

‐mercaptoethanol at pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of butyrate‐induced production of a mannose‐6‐phosphorylated therapeutic enzyme using parallel bioreactors

Madhavarao

Agarabi

Wong

et al. 2014

Biotech and App Biochem

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Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The β-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased β-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in β-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased β-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

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“…2B). β-glucuronidases have molecular weights between 69 to 300 kDa and monomeric and tetrameric forms are found in mammals and bacteria [15,16,17]. The enzyme found in Pomacea eggs is different from those already described because it was shown to be a dimeric protein with an estimated Mr of 180 kDa, being probably two identical subunits single protein band of ~90 kDa,.…”

Section: Resultsmentioning

confidence: 82%

“…The β-glucuronidases found in bacteria are basic enzymes with an optimum activity at pH 5.0 to 7.5 [20,17] and those found in mammalians are acid enzymes with high activity at pH 3.0 to 5.6 [21,22]. The β-glucuronidases isolated from Pomacea eggs had an optimum pH similar those detected in mammals, suggesting lysosomal enzymes.…”

Section: Resultsmentioning

confidence: 91%

Purification and Characterization of a β-Glucuronidase Present During Embryogenesis of the Mollusk Pomacea sp.

Pereira¹,

Cruz²,

Albuquerque³

et al. 2005

PPL

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A beta-glucuronidase was purified from Pomacea sp. eggs by ammonium sulfate fractionation, DEAE-BioGel and Heparin-Sepharose chromatographies. This enzyme showed a Mr 180 kDa, with subunits of 90 kDa. The kinetic parameters were: pH 4.0, temperature 60 degrees C, Km 2.7 x 10(-6) and Vmax 15.3 microM/h, activator Mg+2, and inhibitor: lactone of D-saccharic acid. beta-glucuronidase is an exoglucuronidase involved in glycosaminoglycans metabolism with kinetics parameters similar to those found in mammals.

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Expression and Purification of Escherichia coli β-Glucuronidase

Cited by 10 publications

References 15 publications

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Evaluation of butyrate‐induced production of a mannose‐6‐phosphorylated therapeutic enzyme using parallel bioreactors

Purification and Characterization of a β-Glucuronidase Present During Embryogenesis of the Mollusk Pomacea sp.

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Expression and Purification of Escherichia coli β-Glucuronidase

Cited by 10 publications

References 15 publications

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Evaluation of butyrate‐induced production of a mannose‐6‐phosphorylated therapeutic enzyme using parallel bioreactors

Purification and Characterization of a &#946;-Glucuronidase Present During Embryogenesis of the Mollusk Pomacea sp.

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Purification and Characterization of a β-Glucuronidase Present During Embryogenesis of the Mollusk Pomacea sp.