Characterization of amylomaltase from Thermus filiformis and the increase in alkaline and thermo-stability by E27R substitution

被引：23

作者：

Kaewpathomsri, Piriya ^{[1
]}

Takahashi, Yui ^{[2
]}

Nakamura, Shigeyoshi ^{[3
]}

Kaulpiboon, Jarunee ^{[4
]}

Kidokoro, Shun-ichi ^{[3
]}

Murakami, Shuichiro ^{[2
]}

Krusong, Kuakarun ^{[1
]}

Pongsawasdi, Piamsook ^{[1
]}

机构：

[1] Chulalongkorn Univ, Dept Biochem, Starch & Cyclodextrin Res Unit, Fac Sci, Bangkok 10330, Thailand

[2] Meiji Univ, Grad Sch Agr, Dept Agr Chem, Tama Ku, Kawasaki, Kanagawa 2148571, Japan

[3] Nagaoka Univ Technol, Dept Bioengn, Nagaoka, Niigata 9402188, Japan

[4] Thammasat Univ, Dept Preclin Sci Biochem, Fac Med, Pathum Thani 12120, Thailand

来源：

PROCESS BIOCHEMISTRY | 2015年 / 50卷 / 11期

关键词：

Large-ring cyclodextrin; Amylomaltase; Alkaline stability; Thermostability; Cyclization; Thermus filiformis; CRYSTAL-STRUCTURE; RATIONAL DESIGN; GENE CLONING; 4-ALPHA-GLUCANOTRANSFERASE; CYCLOAMYLOSE; PURIFICATION; STARCH; EXPRESSION; AQUATICUS; COMPLEX;

D O I：

10.1016/j.procbio.2015.08.006

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Amylomaltase catalyzes the alpha-1,4 glycosyl transfer between oligosaccharides. The amylomaltase gene from Thermus filiformis JCM11600 (TfAM) was cloned, expressed in Escherichia coli and purified to homogeneity. TfAM, a member of glycoside hydrolase family 77, encoded the polypeptide of 485 amino acid residues, the shortest among Thermus amylomaltases, with a calculated molecular mass of 55.47 kDa and pI of 5.11. Highest disproportionation activity occurred with maltotriose substrate at pH 6.5 and 60 degrees C to produce linear oligosaccharides. However, highest cyclization activity was observed at pH 5.0 and 70 degrees C, resulting in large-ring cyclodextrins with CD22 as the smallest and CD24-CD29 as principle products. TfAM lost 80% of its disproportionation activity after incubation for 2 h at pH 9.0 or 1 h at 90 degrees C. Meanwhile, E27R-TfAM mutant, forming an Arg cluster (R27-R30-R31-R34) on the enzyme surface, showed a significant increase in stability at these extreme pH and temperature and a shift toward higher pH and temperature optima in cyclization reaction. Conformational change of the mutated enzyme at pH 9.0 and temperature above 350K were observed through the circular dichroism spectra and the thermal transition profiles, respectively. (C) 2015 Elsevier Ltd. All rights reserved.

引用

页码：1814 / 1824

页数：11

共 63 条

[1]

Alam Feroz, 2009, Agriculturae Conspectus Scientificus, V74, P45

[2] Optical mapping and its potential for large-scale sequencing projects [J].

Aston, C ;

Mishra, B ;

Schwartz, DC .

TRENDS IN BIOTECHNOLOGY, 1999, 17 (07) :297-302

[3]

Bang BY, 2006, J MICROBIOL BIOTECHN, V16, P1809

[4] Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre [J].

Bennett-Lovsey, Riccardo M. ;

Herbert, Alex D. ;

Sternberg, Michael J. E. ;

Kelley, Lawrence A. .

PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 2008, 70 (03) :611-625

[5] A cycloamylose-forming hyperthermostable 4-α-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli [J].

Bhuiyan, SH ;

Kitaoka, M ;

Hayashi, K .

JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 2003, 22 (1-2) :45-53

[6]

BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3

[7]

Da Costa M S., 2001, Bergey's Manual of Systematic Bacteriology, V1, P404

[8]

Da-Nian L., 2008, J APPL POLYM SCI, V109, P3733, DOI DOI 10.1002/APP.28612

[9] Structure-based rational design and introduction of arginines on the surface of an alkaline α-amylase from Alkalimonas amylolytica for improved thermostability [J].

Deng, Zhuangmei ;

Yang, Haiquan ;

Shin, Hyun-dong ;

Li, Jianghua ;

Liu, Long .

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 2014, 98 (21) :8937-8945

[10]

Edelstein S.J., 1991, PROTEIN METHODS, P107

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