Journal of Microbiology

Journal Article

Biochemical Characterization of Chitinase 2 Expressed during the Autolytic Phase of the Inky Cap, Coprinellus congregatus: Yuri Kang , Hyewon Kim , Hyoung T. Choi; J. Microbiol. 2013;51(2):189-193. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2535-9

Abstract: Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside was used as the substrate. The Km value and Vmax for the substrate A, 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min-1unit-1, respectively.

Research Support, Non-U.S. Gov'ts

Induction of Growth Phase-Specific Autolysis in Bacillus subtilis 168 by Growth Inhibitors: Jin-Kyo Chung , Hyun Ee Yoon , Ha Chul Shin , Eun-Young Choi , Woo-Hyeon Byeon; J. Microbiol. 2009;47(1):50-59. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0256-2

Abstract: Growth phase-specific autolysis of Bacillus subtilis by inhibitors of membrane permeability, inhibitors of macromolecule biosynthesis, inhibitors of cell wall biosynthesis and detergents were tested and characterized in glucose limited liquid medium. The minimum autolysis induction concentration (MAIC) of test compounds, which was at least 1/20th lower than the conventional autolysis induction concentration, induced autolysis only for cells at the glucose exhaustion point (diauxic point) of the growth phase, while it was not induced for cells at pre- and post-diauxic points. Inhibitors of macromolecule synthesis that are not known for inducing autolysis, such as chloramphenicol, rifampicin, nalidixic acid, and detergents, also induced specific autolysis. Two types of autolysis corresponding to the concentrations of compounds are distinguished: concentration-sensitive and concentration-insensitive types.

Development of a Simple Cell Lysis Method for Recombinant DNA Using Bacteriophage Lambda Lysis Genes: Boyun Jang , Yuna Jung , Dongbin Lim; J. Microbiol. 2007;45(6):593-596.; DOI: https://doi.org/2602 [pii]

Abstract: In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

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