Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase

1994 ◽  
Vol 5 (6) ◽  
pp. 660-665 ◽  
Author(s):  
Yasushi Tamada ◽  
Barbara A. Swanson ◽  
Abolfazl Arabshahi ◽  
Perry A. Frey
Keyword(s):  
Fusion Enzyme ◽  
Bifunctional Fusion Enzyme

scholarly journals Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

2000 ◽  
Vol 66 (6) ◽  
pp. 2484-2490 ◽  
Author(s):  
Hak Soo Seo ◽  
Yeon Jong Koo ◽  
Jae Yun Lim ◽  
Jong Tae Song ◽  
Chung Ho Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Sequential Reaction ◽  
E Coli ◽  
Fusion Enzyme ◽  
Bifunctional Fusion Enzyme ◽  
Sequential Reactions

ABSTRACT To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The Km values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, thek cat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k cat/Km ). The Km and k cat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-β-d-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.

In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris

Biologia ◽  
2012 ◽  
Vol 67 (4) ◽  
Author(s):  
Jiayun Qiao ◽  
Yunhe Cao
Keyword(s):  
Bacillus Subtilis ◽  
Pichia Pastoris ◽  
Fusion Protein ◽  
Fusion Expression ◽  
Chimeric Genes ◽  
Fusion Enzyme ◽  
Fusion Enzymes ◽  
Aspergillus Sulphureus

AbstractTwo chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Kn higher kcat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.

scholarly journals A Novel Bifunctional Fusion Enzyme Catalyzing Ethylene Synthesis via 1-Aminocyclopropane1-carboxylic Acid

1996 ◽  
Vol 271 (42) ◽  
pp. 25738-25741 ◽  
Author(s):  
Ning Li ◽  
Xiang Ning Jiang ◽  
Guo Ping Cai ◽  
Shang Fa Yang
Keyword(s):  
Carboxylic Acid ◽  
Ethylene Synthesis ◽  
Fusion Enzyme ◽  
Bifunctional Fusion Enzyme

scholarly journals Characterization of the  -2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme

Glycobiology ◽  
2007 ◽  
Vol 18 (2) ◽  
pp. 177-186 ◽  
Author(s):  
L. M Willis ◽  
M. Gilbert ◽  
M.-F. Karwaski ◽  
M.-C. Blanchard ◽  
W. W Wakarchuk
Keyword(s):  
Neisseria Meningitidis ◽  
Fusion Enzyme
Sign in / Sign up

Export Citation Format

Share Document