An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities |
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Authors: | Xiaolei Li Dan Li Kwan-Hwa Park |
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Institution: | 1. Key Laboratory of Agricultural Products Processing in Jilin Provincial Universities, Changchun University, Satellite Road 6543, Changchun, 130022, People’s Republic of China 2. Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611, USA 3. Department of Foodservice Management and Nutrition, Sangmyung University, 7 Hongjidong, Jongnogu, Seoul, 110-743, South Korea
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Abstract: | A gene encoding an amylopullulanase of the glycosyl hydrolase (GH) family 57 from Staphylothermus marinus (SMApu) was heterologously expressed in Escherichia coli. SMApu consisted of 639 amino acids with a molecular mass of 75.3 kDa. It only showed maximal amino acid identity of 17.1 % with that of Pyrococcus furiosus amylopullulanase in all identified amylases. Not like previously reported amylopullulanases, SMApu has no signal peptide but contains a continuous GH57N_Apu domain. It had the highest catalytic efficiency toward pullulan (k cat/K m , 342.34 s?1?mL?mg?1) and was extremely thermostable with maximal pullulan-degrading activity (42.1 U/mg) at 105 °C and pH?5.0 and a half-life of 50 min at 100 °C. Its activity increased to 116 % in the presence of 5 mM CaCl2. SMApu could also degrade cyclodextrins, which are resistant to the other amylopullulanases. The initial hydrolytic products from pullulan, γ-CD, and 6-O-maltooligosyl-β-CD were 6)-α-d-Glcp-(1?→?4)-α-d-Glcp-(1?→?4)-α-d-Glcp-(1→]n, maltooctaose, and single maltooligosaccharide plus β-CD, respectively. The final hydrolytic products from above-mentioned substrates were maltose and glucose. These results confirm that SMApu is a novel amylopullulanase of the family GH57 possessing the cyclodextrin-degrading activity of cyclomaltodextrinase. |
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