Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity

• Published in: Applied microbiology and biotechnology Vol. 91; no. 2; pp. 329 - 339
• Main Authors: Kim, Young-Min, Shimizu, Ryoko, Nakai, Hiroyuki, Mori, Haruhide, Okuyama, Masayuki, Kang, Min-Sun, Fujimoto, Zui, Funane, Kazumi, Kim, Doman, Kimura, Atsuo
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.07.2011; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Amino acids; Analysis; Bacteria; Binding sites; Biocatalysis; Biochemical analysis; Biological and medical sciences; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Biotechnology - methods; Catalytic Domain; Dextranase - chemistry; Dextranase - genetics; Dextranase - isolation & purification; Dextranase - metabolism; E coli; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - classification; Glycoside Hydrolases - metabolism; Hydrolysis; Kinetics; Life Sciences; Microbial Genetics and Genomics; Microbiology; Molecular Sequence Data; Peptides; Proteins; Proteomics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Deletion; Streptococcus mutans - enzymology; Streptococcus mutans - genetics; Studies; Substrate Specificity; Endo-dextranase; Limited proteolysis; Glycoside hydrolase family 66; Truncation; Enzyme; Dextranase; Streptococcus mutans; Glycosylases; Streptococcaceae; Proteolysis; Glycosidases; Bacteria; Hydrolases; Micrococcales; Glycoside
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3201-y

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.