Pectin methyl esterase from Aspergillus aculeatus: Expression cloning in yeast and characterization of the recombinant enzyme
Pectin methyl esterase from Aspergillus aculeatus: Expression cloning in yeast and characterization of the recombinant enzyme
复制标题
DOI：
10.1042/bj3190705
复制
发表时间：
1996-11-01
影响因子：
4.1
通讯作者：
Kauppinen, S
中科院分区：
文献类型：
--
作者：
Christgau, S;Kofod, LV;Kauppinen, S
通过酵母表达克隆的方法，从丝状真菌棘孢曲霉（Aspergillus aculeatus）中分离到17个编码果胶甲酯酶I（PME I）的全长cDNA。在含有高度酯化果胶的琼脂平板上鉴定表达功能性PME I的酵母菌落，并分离编码PME I的cDNA。推导的PME I的氨基酸序列与尼日尔曲霉PME高度相似（74%同一性）。将编码PME I的全长cDNA克隆到曲霉表达载体中，并转化到Aspergillusplasma中用于异源表达、纯化和鉴定重组酶。重组PME I的分子量为36.2 kDa，等电点为pH3.8，最适pH为4.6，最适温度为45 ℃。PME I是从A. Aculeatus培养物上清液并进行氨基酸测序。肽段序列由138个氨基酸组成，与推测的PME I序列完全一致。重组PME I和真PME I均为糖基化，但聚糖组成不同。PME I能够去除高度甲基化果胶中75- 85%的甲基，但不能去除乙酰化多糖中的乙酰基。当酶与多聚半乳糖醛酸酶一起添加到果胶中时，观察到快速解聚。通过比较，单独的多聚半乳糖醛酸酶显示出非常有限的甲基化底物降解。这表明PME I在植物细胞壁果胶的降解中与多聚半乳糖醛酸酶协同作用。
Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74 % identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence, Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85 % of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.