Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Japan.
Biochimie. 2011 Oct;93(10):1720-30. doi: 10.1016/j.biochi.2011.06.004. Epub 2011 Jun 12.
Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5'- and 3'-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.
草食性海洋腹足纲动物（如鲍鱼和海兔）以褐藻为主要食物，并在其消化液中用褐藻胶裂解酶（EC 4.2.2.3）将膳食中的褐藻酸盐降解。迄今为止，已对来自鲍科物种（如鲍鱼）的褐藻胶裂解酶进行了很好的表征，并且通过对其一级结构分析，将鲍鱼酶归类为多糖裂解酶家族 14（PL-14）。然而，其他腹足纲动物的酶尚未得到很好的研究，目前仅提供部分氨基酸序列。为了提高对腹足纲褐藻胶裂解酶的一级结构和催化残基的了解，我们从 Aplysiidae 物种 Aplysia kurodai 克隆了编码褐藻胶裂解酶的 cDNA，并通过定点诱变评估了其催化重要残基。通过 PCR 从 A. kurodai 肝胰腺 cDNA 扩增了褐藻胶裂解酶 cDNA 片段，然后进行 5'和 3'RACE。最终克隆的 cDNA 由 1313bp 组成，编码 295 个氨基酸的 AkAly30 序列。推导的序列包含一个起始甲硫氨酸、一个用于分泌的假定信号肽（18 个残基）、一个前肽样区（9 个残基）和一个成熟的 AkAly30 结构域（267 个残基），与鲍鱼褐藻胶裂解酶的氨基酸同一性约为 40%。构建了用于重组 AkAly30（recAkAly30）的大肠杆菌 BL21(DE3)-pCold I 表达系统，并进行了定点诱变，以评估已在鲍鱼和绿藻病毒 PL-14 酶中提出的催化重要氨基酸残基。用丙氨酸和/或苯丙氨酸替换 recAkAly30 的 K99、S126、R128、Y140 和 Y142 极大地降低了其活性，就像鲍鱼和/或绿藻病毒酶的情况一样。然而，对于绿藻病毒酶在 pH 10.0 下表现出活性至关重要的 H213，在 AkAly30 中最初被 N120 取代。在 recAkAly30 中，将 N120 反向替换为 His 使 pH 10.0 下的活性从 8U/mg 增加到 93U/mg；然而，pH 7.0 下的活性水平，即 774.8U/mg，仍然远高于 pH 10.0 下的活性。这表明与 vAL-1 的 H213 不同，N120 与 AkAly30 的 pH 依赖性无关。
