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Englert, Markus,Xia, Shuangluo,Okada, Chiaki,Nakamura, Akiyoshi,Tanavde, Ved,Yao, Min,Eom, Soo Hyun,Konigsberg, William H.,Sö,ll, Dieter,Wang, Jimin Proceedings of the National Academy of Sciences 2012 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.109 No.38
<P>The RtcB protein has recently been identified as a 3′-phosphate RNA ligase that directly joins an RNA strand ending with a 2′,3′-cyclic phosphate to the 5′-hydroxyl group of another RNA strand in a GTP/Mn<SUP>2+</SUP>-dependent reaction. Here, we report two crystal structures of <I>Pyrococcus horikoshii</I> RNA-splicing ligase RtcB in complex with Mn<SUP>2+</SUP> alone (RtcB/ Mn<SUP>2+</SUP>) and together with a covalently bound GMP (RtcB-GMP/Mn<SUP>2+</SUP>). The RtcB/ Mn<SUP>2+</SUP> structure (at 1.6 Å resolution) shows two Mn<SUP>2+</SUP> ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn<SUP>2+</SUP> complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn<SUP>2+</SUP> ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (<I>i</I>) guanylylation of the enzyme, (<I>ii</I>) guanylyl-transfer to the RNA substrate, and (<I>iii</I>) overall ligation. These results demonstrate that the enzyme’s substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn<SUP>2+</SUP> active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3′-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5′-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.</P>
Sohail Rasool Lone,Vimal Kumar,Jeffrey R. Seay,Derek L. Englert,Hyun Tae Hwang 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.5
With the rapid growth of pharmaceutical and biotechnology industry, stirred tank bioreactors have received much attention due to simple design, easy control of operating conditions, and low operating cost. In the development of commercial processes, however, a transition from laboratory to industrial scale faces great challenges because many properties related to size change nonlinearly as a system increases. In this context, along with an understanding of fluid dynamics, application of an efficient method for scale-up is critical for designing successful industrial process. Particularly in cell cultivation processes, it is important to evaluate the oxygen transfer and viscous properties of liquid medium. In the present study, the effect of various key operating variables such as agitation rate and aeration rate, impeller diameter, and bioreactor working volume for different impellers on the volumetric mass transfer coefficient (kLa) have been investigated in a stirred tank bioreactor for cultivating Escherichia coli BL21. It was found that the kLa tends to increase with the operating variables except the bioreactor working volume. Among the tested impellers, the pitched blade was observed to be most promising because of relatively higher kLa but less shear force owing to its low power number. It was also found that the liquid medium with E. coli behaves as a Newtonian liquid. Compared to conventional designs of Rushton turbines, dislocated Rushton turbine was found to deliver higher kLa. Finally, using dimensional analysis, the kLa for different impeller configurations was correlated in the form of dimensionless groups, suggesting that this approach can be used for predicting kLa in different scales of stirred tank bioreactors.