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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2014/11/28 09:30:02」(JST)
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English Journal
- Protein engineering of α2,3/2,6-sialyltransferase to improve the yield and productivity of in vitro sialyllactose synthesis.
- Choi YH, Kim JH, Park JH, Lee N, Kim DH, Jang KS, Park IH, Kim BG.Author information Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics.AbstractIn the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galβ1,4Glc (3'-SL) and Neu5Ac(α2,6)Galβ1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Pasteurella multocida α2,3-sialyltransferase (α2,3PST) forms a by-product to a certain extent, owing to its multifunctional activity at pH below 7.0, and Photobacterium damselae α2,6-sialyltransferase (α2,6PdST) shows relatively low ST activity. In this study, α2,3PST and α2,6PdST were successfully engineered using a hybrid approach that combines rational design with site-saturation mutagenesis. Narrowly focused on the substrate-binding pocket of the STs, putative functional residues were selected by multiple sequence alignment and alanine scanning, and subsequently subjected to site-saturation mutagenesis. In the case of α2,3PST, R313N single mutation improved its activity slightly (by a factor of 1.5), and further improvement was obtained by making the double mutants (R313N/T265S and R313H/T265S) resulting in an overall 2-fold improvement in its specific α2,3 ST activity, which is mainly caused by the increase in kcat. It was revealed that the R313 mutations to N, D, Y, H or T greatly reduced the α2,6 ST side-reaction activity of α2,3PST at below pH 7.0. In the case of α2,6PdST, single-mutation L433S/T and double-mutation I411T/L433T exhibited 3- and 5-fold enhancement of the α2,6 ST-specific activity compared with the wild-type, respectively, via increase in kcat values. Our results show a very good model system for enhancing ST activity and demonstrate that the generated mutants could be used efficiently for the mass production of 3'-SL and 6'-SL with enhanced productivity and yield.
- Glycobiology.Glycobiology.2014 Feb;24(2):159-69. doi: 10.1093/glycob/cwt092. Epub 2013 Oct 18.
- In the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galβ1,4Glc (3'-SL) and Neu5Ac(α2,6)Galβ1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Past
- PMID 24142961
- A Pasteurella multocida sialyltransferase displaying dual trans-sialidase activities for production of 3'-sialyl and 6'-sialyl glycans.
- Guo Y1, Jers C1, Meyer AS1, Arnous A1, Li H2, Kirpekar F2, Mikkelsen JD3.Author information 1Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800 Kgs. Lyngby, Denmark.2Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.3Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800 Kgs. Lyngby, Denmark. Electronic address: jdm@kt.dtu.dk.AbstractThis study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose as the acceptor, resulting in production of both 3'-sialyllactose and 6'-sialyllactose. This is the first study reporting α-2,6-trans-sialidase activity of this sialyltransferase (EC 2.4.99.1 and 2.4.99.4). A response surface design was used to evaluate the effects of three reaction parameters (pH, temperature, and lactose concentration) on enzymatic production of 3'- and 6'-sialyllactoses using 5% (w/v) casein glycomacropeptide (equivalent to 9mM bound sialic acid) as the donor. The maximum yield of 3'-sialyllactose (2.75±0.35mM) was achieved at a reaction condition with pH 6.4, 40°C, 100mM lactose after 6h; and the largest concentration of 6'-sialyllactose (3.33±0.38mM) was achieved under a condition with pH 5.4, 40°C, 100mM lactose after 8h. 6'-sialyllactose was presumably formed from α-2,3 bound sialic acid in the casein glycomacropeptide as well as from 3'-sialyllactose produced in the reaction. The kcat/Km value for the enzyme using 3'-sialyllactose as the donor for 6'-sialyllactose synthesis at pH 5.4 and 40°C was determined to be 23.22±0.7M(-1)s(-1). Moreover, the enzyme was capable of catalyzing the synthesis of both 3'- and 6'-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors.
- Journal of biotechnology.J Biotechnol.2014 Jan 20;170:60-7. doi: 10.1016/j.jbiotec.2013.11.013. Epub 2013 Nov 27.
- This study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose as the
- PMID 24291191
- Control of pili and sialyltransferase expression in Neisseria gonorrhoeae is mediated by the transcriptional regulator CrgA.
- Matthias KA, Rest RF.Author information Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, USA.AbstractContact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific subset of genes that CrgA targets has been debated. Although the majority of these constitute virulence genes, suggesting that CrgA is important in pathogenesis, no study to date has examined the effects of CrgA in Neisseria gonorrhoeae. In this report, we generated a knockout mutant of crgA (ΔcrgA) in the serum sensitive gonococcal strain F62. crgA deletion resulted in a reduction in the transcript and protein levels of the primary pilin component pilE via mechanisms that were both contact-dependent and -independent. In contrast, ΔcrgA overexpressed the main determinant of serum resistance in F62, lipooligosaccharide sialyltransferase (Lst). CrgA-mediated lst repression was direct as both recombinant and native CrgA bound to the lst promoter at multiple locations in EMSA and ChIP assays, respectively. The increase in Lst levels associated with crgA deletion correlated with enhanced protection against killing by normal human serum. These data suggest a role for CrgA in virulence regulation during both cell-adherence and planktonic growth.
- Molecular microbiology.Mol Microbiol.2014 Jan 17. doi: 10.1111/mmi.12522. [Epub ahead of print]
- Contact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific
- PMID 24433334
Japanese Journal
- Ganglioside, Disialosyl Globopentaosylceramide (DSGb5), Enhances the Migration of Renal Cell Carcinoma Cells
- Kawasaki Yoshihide,Ito Akihiro,Kakoi Narihiko,Shimada Shuichi,Itoh Jun,Mitsuzuka Koji,Arai Yoichi
- The Tohoku Journal of Experimental Medicine 236(1), 1-7, 2015
- … DSGb5 is synthesized by GalNAc α2,6-sialyltransferase VI (ST6GalNAcVI) and is expressed on the surface of RCC cells. …
- NAID 130005061506
- TFE3 Is a bHLH-ZIP-type Transcription Factor that Regulates the Mammalian Golgi Stress Response
- Taniguchi Mai,Nadanaka Satomi,Tanakura Soichiro,Sawaguchi Shogo,Midori Sachiko,Kawai Yumeto,Yamaguchi Shogo,Shimada Yui,Nakamura Yuki,Matsumura Yasuyo,Fujita Natsumi,Araki Naoko,Yamamoto Mayu,Oku Masaya,Wakabayashi Sadao,Kitagawa Hiroshi,Yoshida Hiderou
- Cell Structure and Function 40(1), 13-30, 2015
- The Golgi stress response is a mechanism by which, under conditions of insufficient Golgi function (Golgi stress), the transcription of Golgi-related genes is upregulated through an enhancer, the Golg …
- NAID 130004885864
- The Loss of Luteal Progesterone Production in Women Is Associated With a Galectin Switch via alpha 2,6-Sialylation of Glycoconjugates
- Nio-Kobayashi Junko,Boswell Lyndsey,Amano Maho,Iwanaga Toshihiko,Duncan W. Colin
- Journal of clinical endocrinology & metabolism 99(12), 4616-4624, 2014-12
- … The effects of hCG and prostaglandin E-2 on the expression of galectins and an alpha 2,6-sialyltransferase 1 (ST6GAL1) in granulosa lutein cells were analyzed in vitro. …
- NAID 120005553690
Related Links
- Sialyltransferases are enzymes that transfer sialic acid to nascent oligosaccharide.Each sialyltransferase is specific for a particular sugar substrate. Sialyltransferases add sialic acid to the terminal portions of the sialylated glycolipids ...
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