Department of Biochemistry, Kangwon National University, Chunchon 200-701, Republic of Korea.
J Microbiol. 2013 Apr;51(2):189-93. doi: 10.1007/s12275-013-2535-9. Epub 2013 Apr 27.
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 K m value and V max for the substrate A, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min(-1)unit(-1), respectively.
真菌细胞壁由各种葡聚糖和几丁质组成。鬼伞属的墨汁鬼伞在 25°C 下、15 小时光照/9 小时黑暗的条件下产生蘑菇，然后蘑菇迅速自溶，产生黑色液滴，显微镜下检测不到细胞壁。使用快速扩增 cDNA 末端聚合酶链反应技术，成功地从成熟蘑菇组织中克隆了墨汁鬼伞的几丁质酶 cDNA，其由 1622 个核苷酸（chi2）组成。推断的 Chi2 498 个氨基酸序列在其他真菌几丁质酶家族 18 酶中具有保守的催化结构域。Chi2 酶从毕赤酵母表达系统中纯化，其估计分子量为 68 kDa。当 4-硝基苯基 N，N'-二乙酰基-β-D-壳二糖苷作为底物时，Chi2 的最适 pH 和温度分别为 pH 4.0 和 35°C。底物 A，4-硝基苯基 N，N'-二乙酰基-β-D-壳二糖苷的 K m 值和 V max 值分别为 0.175 mM 和 0.16 OD min(-1)unit(-1)。
