Truncation of N- and C-terminal regions of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> dextranase enhances catalytic activity |
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Authors: | Young-Min Kim Ryoko Shimizu Hiroyuki Nakai Haruhide Mori Masayuki Okuyama Min-Sun Kang Zui Fujimoto Kazumi Funane Doman Kim Atsuo Kimura |
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Institution: | (1) Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060–8589, Japan;(2) Eco-Friendly Biomaterial Research Center and AI control Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, 580-185, Republic of Korea;(3) Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan;(4) National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan;(5) National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba 305-8642, Japan;(6) School of Biological Sciences and Technology, Chonnam National University, Gwangju, 500-757, Republic of Korea |
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Abstract: | Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66
Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90
was proteolytically degraded to more than seven polypeptides (23–70 kDa) during long storage. The protease-insensitive protein
was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from
the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable
region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid
of C-VR (TM-NCGΔ) or N-VR/C-VR (TM-ΔCGΔ) were catalytically active, thereby indicating that N-VR and C-VR were not essential
for the catalytic activity. TM-ΔCGΔ did not accept any further protease-degradation during long storage. TM-NCGΔ and TM-ΔCGΔ
enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site. |
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