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MOLECULAR CLONING OF TWO CHITINASE GENES FROM SERRATIA MARCESCENS K. AND EXPRESSION OF THE GENES IN E. COLI

Ha, Mi-Sook (Department of Biochemistry, Gyeongsang National University) , Gal, Sang-Wan (Plant Molecular Biology and Biotechnology Research Center) , Kim, Cha-Young (Plant Molecular Biology and Biotechnology Research Center) , Koo, Ja-Chun (Plant Molecular Biology and Biotechnology Research Center) , Bae, Chang-Gyu (Department of Biochemistry, Gyeongsang National University) , Choi, Young-Ju (Plant Molecular Biology and Biotechnology Research Center) , Chun, Hyun-Jin (Department of Biochemistry, Gyeongsang National University) , Lee, Sang-Yeol (Plant Molecular Biology and Biotechnology Research Center, Department of Biochemistry, Gyeongsang National University) , Bahk, Jeong-Dong (Plant Molecular Biology and Biotechnology Research Center, Department of Biochemistry, Gyeongsang National University) , Cho, Moo-Je (Plant Molecular Biology and Biotechnology Research Center, Department of Biochemistry, Gyeongsang National University)

Plant molecular biology and biotechnology research center Annual report, 1994, 1994, 257-269

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To investigate the regulatory mechanisms and enzymatic characteristics of chitinolytic activity in Serattia marcescens k, two genes encoding 52kD 2kD chitinase were isolated from a cosmid library prepared from S. marcescens genomic DNA and expressed in Escherichia coli. Chitinase activity of the ins...
To investigate the regulatory mechanisms and enzymatic characteristics of chitinolytic activity in Serattia marcescens k, two genes encoding 52kD 2kD chitinase were isolated from a cosmid library prepared from S. marcescens genomic DNA and expressed in Escherichia coli. Chitinase activity of the insert bearing the cloned was detected on chitin medium and confirmed by unbinding profiles of the fluorescent chitin binding reagent (Calcofluor White M2R) on SDS-PAGE gel containing glycol chitin. The chitinases were purified chitin affinity column chromatography from culture supernatant of E. coli transformants. Nucleotide and peptide sequences of the two chitinase were determined. The 52kD chitinase gene was consisted of 2563bp of open reading frame encoding 521 amino acids. The 52kD chitinase showed 97.3% sequence similarity to 52kD chitinase reported by Mark et al (1989). The leader sequence of the enzyme was not detected in the N-terminal region of the purified 52kD chitinase. The 21kD chitinase gene was located to 1504 bp downstream from the termination codon of 52kD and it had 666bp of open reading frame which encoded 222 amino acids. The N-terminal amino acid sequence of the 25kD chitinase determined from purified protein was STRKAVIGYYFIPTNQINNY and that of 21kD isozyme was HGYVESPASRPASRAYQCKLQLNTQXGXVQYEPQ. The 52kD chitinase cleaved chitohexaose to predominantly N,N'-diacetylchitobiose and a trace amount of N-acetylglucosamine. However 21kD isozyme produced only N,N'-diacetylchitobiose.order. The optimum pH temperature of 52kD chitinase were 5.0-6.0 and 50℃, those of 2kD chitinase were pH 6.0, 65℃, respectively.
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Ⅰ. Introduction = 257
Ⅱ. Main Contents = 257
Ⅲ. Summary = 268
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Ⅰ. Introduction = 257
Ⅱ. Main Contents = 257
Ⅲ. Summary = 268