caa a22 4500 47574411X CHVBK 20180406123508.0 cr unu---uuuuu 170329e20001001xx s 000 0 eng 10.1023/A:1007147214796 doi (NATIONALLICENCE)springer-10.1023/A:1007147214796 Analysis of Essential Leucine Residue for Catalytic Activity of Novel Thermostable Chitosanase by Site-Directed Mutagenesis [Elektronische Daten] [Ho Yoon, Sung Yang, Hee Kim, Hye Kim, Dong Shin, Bum Hong, Hong Cho] Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 → Arg and Leu64 → Gln mutations were essentially inactive and kinetic parameters such as V max and k cat were approximately 1/107 of those of the wild-type enzyme. The Asp98 → Asn mutation did not affect the K m value significantly, but decreased k cat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 srarr; Glu mutation affected neither K m nor k cat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2. Plenum Publishing Corporation, 2000 Thermostable chitosanase nationallicence Bacillus coagulans CK108 nationallicence site-directed mutagenesis nationallicence leucine residue nationallicence catalytic activity nationallicence Yoon Ho Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut Yang Sung Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut Kim Hee Department of Food Additives, Korea Food and Administration, 122-202, Seoul, Korea aut Kim Hye Department of Food and Biotechnology, Hanseo University, 352-820, Chungnam, Korea aut Shin Dong Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut Hong Bum Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut Cho Hong Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/7(2000-10-01), 621-630 0277-8033 19:7<621 2000 19 10930 https://doi.org/10.1023/A:1007147214796 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1007147214796 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Yoon Ho Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut NATIONALLICENCE 700 1- Yang Sung Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut NATIONALLICENCE 700 1- Kim Hee Department of Food Additives, Korea Food and Administration, 122-202, Seoul, Korea aut NATIONALLICENCE 700 1- Kim Hye Department of Food and Biotechnology, Hanseo University, 352-820, Chungnam, Korea aut NATIONALLICENCE 700 1- Shin Dong Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut NATIONALLICENCE 700 1- Hong Bum Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut NATIONALLICENCE 700 1- Cho Hong Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul, Korea aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/7(2000-10-01), 621-630 0277-8033 19:7<621 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer