Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phyathai Road, Bangkok 10330, Thailand.
Comp Biochem Physiol B Biochem Mol Biol. 2010 Jun;156(2):86-96. doi: 10.1016/j.cbpb.2010.02.007. Epub 2010 Mar 1.
Chitinases are essential enzymes for crustaceans and animal alike for their molting and digestion of foods containing chitin. From the Penaeus monodon EST database, cDNA contigs and singletons for three chitinases, namely PmChi1, 2 and 3, were identified. The complete sequences for the mature PmChi1, 3 and partial PmChi2 were amplified and cloned. The reading frames of PmChi1 and 3 encoded mature proteins of 644 and 468 amino acids with calculated molecular masses of 72.4 and 51.9kDa, respectively. The amino acid sequence comparison among the penaeid chitinases revealed homology around 90%. Therefore, they were grouped together along with those of other crustaceans and insects into three groups separated from those of mammals. PmChi1, 2 and 3 were expressed mainly in hepatopancreas, gill and hepatopancreas, respectively, though small amounts were expressed in other tissues. After molting, only the expression of PmChi2 was down-regulated, while the expression of PmChi1 and 3 was relatively unchanged. The results suggested that the PmChi2 was likely involved in molting while the others might function in the digestion of chitinous foods. The recombinant PmChi1 (rPmChi1) over-produced from Escherichia coli had its optimal pH 5 but it was most stable at neutral pH. Interestingly, the optimal temperature was relatively high at 55 degrees C. Nevertheless, it was stable at lower temperature below 40 degrees C. The rPmChi1 preferentially hydrolyzed the more soluble substrates like partially N-acetylated chitin (PNAC) and colloidal chitin from shrimp shell as compared to the beta-chitin from squid pen.
几丁质酶是甲壳类动物和动物蜕皮和消化含几丁质食物所必需的酶。从斑节对虾 EST 数据库中，鉴定了三个几丁质酶，即 PmChi1、2 和 3 的 cDNA 丛和单核苷酸。成熟的 PmChi1、3 和部分 PmChi2 的完整序列被扩增和克隆。PmChi1 和 3 的阅读框编码成熟蛋白，分别为 644 和 468 个氨基酸，计算的分子量分别为 72.4 和 51.9kDa。在对虾几丁质酶之间的氨基酸序列比较表明同源性约为 90%。因此，它们与其他甲壳类动物和昆虫一起分为三组，与哺乳动物的几丁质酶分开。PmChi1、2 和 3 主要在肝胰腺、鳃和肝胰腺中表达，尽管在其他组织中也有少量表达。蜕皮后，只有 PmChi2 的表达下调，而 PmChi1 和 3 的表达相对不变。结果表明，PmChi2 可能参与蜕皮，而其他几丁质酶可能在几丁质食物的消化中发挥作用。重组 PmChi1（rPmChi1）从大肠杆菌中过度表达，其最佳 pH 值为 5，但在中性 pH 值下最稳定。有趣的是，最佳温度相对较高，为 55 摄氏度。然而，它在低于 40 摄氏度的低温下稳定。与鱿鱼笔的β-几丁质相比，rPmChi1 优先水解更易溶解的底物，如部分 N-乙酰化几丁质（PNAC）和虾壳胶体几丁质。
