Characterization of Purine Nucleoside Phosphorylase from <em>Bacillus subtilis</em> and Application in Enzymic Production of Ribavirin

China Biotechnology

2010, Vol. 30

Issue (12): 53-59 DOI:

Characterization of Purine Nucleoside Phosphorylase from Bacillus subtilis and Application in Enzymic Production of Ribavirin

XIA Jun-gang, HE Kui-fu, XIE Xi-xian, XU Qing-yang, CHEN Ning

College of Biotechnology, Tianjin University of Science and Technology, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin 300457, China

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Abstract

The purine nucleoside phosphorylas (PNP,EC.2.4.2.1) genes deoD and punA which amplified from the Bacillus subtilis168 genome by polymerase chain reaction were identified, cloned and expressed in E. coli XL-Blue, respectively. Recombinant purine nucleoside phosphorylases PNP₇₀₂ and PNP₈₁₆ were purified by Ni⁺-NTA column, and several characteristics were determined. The results revealed that PNP₇₀₂and PNP₈₁₆ both have the same optimal temperature (60℃) and pH (7~8). Enzymic kinetics experiment showed that the catalytic efficiency (K_cat/K_m) of PNP₈₁₆is 11.12 times higher than that of PNP₇₀₂ toward inosine. Compared with PNP₈₁₆, PNP₇₀₂ has broader substrate specificity. The engineering strain XL-Blue (pPNP₈₁₆) has much higher catalytic activity than the XL-Blue (pPNP₇₀₂) in enzymatic synthesizing of the nucleoside antiviral drugs ribavirin, indicating that the low molecular weight homologous trimer PNP derived from microorganisms has the same or higher value in the microbial enzymatic synthesis of nucleoside drugs and intermediates.

Key words： Purine nucleoside phosphorylase Enzymatic properties Substrate-specificity Ribavirin Enzymic

Received: 08 September 2010 Published: 25 December 2010

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	XIA Jun-gang
	HE Kui-fu
	XIE Xi-xian
	XU Qing-yang
	CHEN Ning

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XIA Jun-gang, HE Kui-fu, XIE Xi-xian, XU Qing-yang, CHEN Ning. Characterization of Purine Nucleoside Phosphorylase from Bacillus subtilis and Application in Enzymic Production of Ribavirin. China Biotechnology, 2010, 30(12): 53-59.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2010/V30/I12/53


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