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Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli.

ABSTRACT: Uricase as an important healthcare-related protein is extensively used in the treatment of tumor lysis syndrome and in the manufacture of serum uric-acid diagnostic kits. In this study, a gene of a new thermostable uricase (KmUOX) was cloned from thermotolerant yeast Kluyveromyces marxianus. The uricase was fused with a self-cleaving intein and cellulose-binding affinity tag and expressed in Escherichia coli BL21 (DE3). Through the binding to inexpensive cellulose and in situ intein cleavage induced by a pH change, tag-free uricase (KmUOX) was efficiently purified with a 77.11% yield via a single-step column purification strategy. This tag-free uricase showed Km, Vmax, and Kcat values of 67.60 µM, 56.35 µM/(min mg), and 32.74 S-1, respectively. Furthermore, this pure uricase was relatively thermostable and retained 79.75% of activity when incubated at 40 °C for 90 h. Thus, this pH-induced self-cleavable intein system combined with a cellulose matrix for affinity chromatography is proven here to be an effective and low-cost method for recombinant-uricase purification. Moreover, the stability of KmUOX makes it useful for clinical applications.

SUBMITTER: Wang B

PROVIDER: S-EPMC6128813 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli.

Wang Bangchun B Luo Laipeng L Wang Dongmei D Ding Rui R Hong Jiong J

3 Biotech 20180907 9

Uricase as an important healthcare-related protein is extensively used in the treatment of tumor lysis syndrome and in the manufacture of serum uric-acid diagnostic kits. In this study, a gene of a new thermostable uricase (KmUOX) was cloned from thermotolerant yeast Kluyveromyces marxianus. The uricase was fused with a self-cleaving intein and cellulose-binding affinity tag and expressed in Escherichia coli BL21 (DE3). Through the binding to inexpensive cellulose and in situ intei ...[more]

PMID: 30221113

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Project description:BackgroundAs a white biotechnological trend, esterases are thought to be among the most active enzymes' classes in biocatalysis and synthesis of industrially importance organic compounds. Esterases are used in many applications such as the manufacture of pharmaceuticals, cosmetics, leather, textile, paper, food, dairy products, detergents, and treatment of some environmental pollutants.ResultsA poly-histidine moiety was added to the C-terminal end of the Geobacillus sp. gene encoding carboxyl esterase (EstB, ac: KJ735452) to facilitate one-step purification. This recombinant protein was successfully expressed in Escherichia coli (E. coli) under control of Lambda promoter (λ). An open reading frame (ORF) of 1500 bps encoding a polypeptide of 499 amino acid residues and a calculated molecular weight (54.7 kD) was identified as carboxyl-esterase B due to its conserved glycine-X-serine-X-glycine motif (G-X-S-X-G) and its high similarity toward other carboxyl esterases, where the 3-D tertiary structure of EstB was determined based on high homology % (94.8) to Est55. The expression was scaled up using 7-L stirred tank bioreactor, where a maximum yield of enzyme was obtained after 3.5 h with SEA 51.76 U/mg protein. The expressed protein was purified until unity using immobilized metal affinity chromatography (IMAC) charged with cobalt and then characterized. The purified enzyme was most active at pH 8.0 and remarkably stable at pH (8-10). Temperature optimum was recorded at 65 °C, and it kept 70% of its activity after 1-h exposure to 60 °C. The active half-live of enzyme was 25 min at 70 °C and a calculated T melting (Tm) at 70 °C. The determined reaction kinetics Michaelis-Menten constant (Km), maximum velocity rate (Vmax), the turnover number (Kcat), and catalytic efficiency (Kcat/Km) of the pure enzyme were found 22.756 mM, 164.47 U/ml (59.6 min-1), and (2.619 mol/ min), respectively.ConclusionCreation of a recombinant 6 × -His estB derived from a thermophile Geobacillus sp. was performed successfully and then overexpressed under λ-promoter. In a bench scale bioreactor, the overexpression was grown up, followed by one-step purification and biochemical characterization. The recorded promising pH and temperature stability properties suggest that this expressed carboxyl esterase could be used in many industrial sectors.

Dataset Information

Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli.

Publications

Efficient purification of a recombinant tag-free thermostable <i>Kluyveromyces marxianus</i> uricase by pH-induced self-cleavage of intein and expression in <i>Escherichia coli</i>.

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