Browsing by Author "Hwang, Long-Chih"

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In Vitro Selection and Characterization of a Novel Zn(II)-Dependent Phosphorothiolate Thiolesterase Ribozyme
(2012-01-10)
Wang, Tzu-Pin
;
Su, Yu-Chih
;
Chen, Yi
;
Liou, Yi-Ming
;
Lin, Kun-Liang
Here we present the in vitro selection of a novel ribozyme specific for Zn(2+)-dependent catalysis on hydrolysis of a phosphorothiolate thiolester bond. The ribozyme, called the TW17 ribozyme, was evolved and selected from an artificial RNA pool covalently linked to a biotin-containing substrate through the phosphorothiolate thiolester bond. The secondary structure for the evolved ribozyme consisted of three major helices and three loops. Biochemical and chemical studies of ribozyme-catalyzed reaction products provided evidence that the ribozyme specifically catalyzes hydrolysis of the phosphorothiolate thiolester linkage. A successful ribozyme construct with active catalysis in trans further supported the determined ribozyme structure and indicated the potential of the ribozyme for multiple-substrate turnover. The ribozyme also requires Zn(2+) and Mg(2+) for maximal catalysis. The TW17 ribozyme, in the presence of Zn(2+) and Mg(2+), conferred a rate enhancement of at least S orders of magnitude when compared to the estimated rate of the uncatalyzed reaction. The ribozyme completely lost catalytic activity in the absence of Zn(2+), like Zn(2+)-dependent protein hydrolases. The discovery and characterization of the TW17 ribozyme suggest additional roles for Zn(2+) in ribozyme catalysts.
Successful preparation and characterization of biotechnological grade agarose from indigenous Gelidium amansii of Taiwan
(2012-03-01)
Wang, Tzu-Pin
;
Chang, Li-Lin
;
Chang, Sheng-Nan
;
Wang, Eng-Chi
;
Hwang, Long-Chih
This paper reports the first successful preparation of biotechnological grade agarose from Gelidium amansii found in Taiwan. The scale-efficiency preparation was achieved by shortening EDTA treatment time through dispersing G. amansii agar in water in the presence of heat and EDTA, removing agaropectin impurity with a heat-compatible and strong-anion exchange resin, and precipitating agarose with a cost-effective isopropanol method. The yield of agarose from prepared G. amansii agar was 11.3%. The acquired agarose has a gel strength of 853 g cm(-2), a sulfate content of 0.14%, a pyruvate content of 1.03%, a degree of electroendosmosis of 0.16 and very limited binding affinity to DNA. The excellent properties of agarose from G. amansii of Taiwan confirm its potential diverse biotechnological applications. This innovative agarose preparation method with the significantly improved scale-efficiency can be modified for large-scale preparation of agarose for use in biotechnological industry and biochemical research. (C) 2011 Elsevier Ltd. All rights reserved.