Molecular cloning and characterization of a new aspartic protease gene from Rhizopus hangchow

Abstract

A new protease (RHAPN) was purified from the culture broth of Rhizopus hangchow IFO4749 by sequential fast protein liquid chromatography procedures. The RHAPN had a molecular weight of about 34 kDa and was active at acidic pH without the presence of divalent metal ion. The inhibition profile and partial peptides sequence indicated that RHAPN might belong to the fungal secreted aspartic protease (SAP) family. The DNA fragment containing part of RHAPN gene was amplified by PCR using degenerate primers designed from the N-terminal peptides sequence and the conserve region of fungal SAPs. This DNA fragment encoded a polypeptide of 296 amino acids and was homologous with rhizopuspepsin III precursor from R. niveus. An efficient method for the preparation of R. oryzae protoplasts was established by using four lytic enzymes in combination, and a shuttle vector containing the A. oryzae ptrA gene as selectable marker was also constructed.

天門冬安酸胜肽分解酵素 (aspartic protease) 為一種蛋白水解酶，被廣泛的應用於食品工業。本研究由商業化生產之 Peptidase R 中純化得到一種蛋白水解酶，命名為RHAPN。經由其部分胜肽序列及其酵素特性分析，發現此酵素屬於真菌胞外天門冬安酸胜肽分解酵素家族 (secreted aspartic protease family, SAP) 之一員。利用 RHAPN 之 N 端序列及真菌 SAPs 之高度保留區設計成退化性引子，可自 Peptidase R 之生產菌 Rhizopus hangchow IFO4749 基因組中選殖出一段約 1 kb 之DNA片段。將預測之內涵子 (intron) 自此序列中去除後，所得到之DNA片段可轉錄出具 296 個氨基酸之胜肽，此胜肽與已發表之 R. nivies rhizopuspepsin III 有 67% 之相同度。經由軟體分析後發現，此基因可能為目前所發現之 Rhizopus spp. SAPs 之祖先。在建立真菌基因表現平台系統方面，目前發現 pyrithiamine 可抑制 R. oryzae 之生長，並已將 pyrithiamine resistant gene (ptrA) 構築入穿梭載體 pskGA 中。此外，本實驗亦成功利用 Novozyme 234、yatalase、chitinase 及 chitosanase 建立 R. oryzae 原生質體之製備方法。