The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger
The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger
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黑曲霉糖蛋白葡糖淀粉酶 G1 的完整氨基酸序列
DOI：
10.1007/bf02907555
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
E. Boel
中科院分区：
文献类型：
--
作者：
B. Svensson;K. Larsen;I. Svendsen;E. Boel
测定了黑曲霉（Aspergillus尼日尔）糖化酶G1（EC 3.2.1.3）的一级结构。用溴化氰、羟胺或S裂解得到G1片段。金黄色葡萄球菌V8蛋白酶。使用DEAE-Sephacel上的离子交换层析、凝胶过滤和Con A-Sepharose上的亲和层析分离所得肽。通过用邻碘异苯甲酸或BNPS-粪臭素切割以及通过用S.金黄色葡萄球菌V8蛋白酶、胰蛋白酶和内切蛋白酶Lys-C。通过上述方法和反相HPLC纯化这些肽。对本发明的片段进行氨基酸测序，并且这允许与胰蛋白酶肽（Carlsberg Res. Commun. 48，517-527（1983）），鉴定了G1中614个氨基酸残基中的574个。对从A.尼日尔总mRNA，能够推导出定位于6个短片段的剩余40个氨基酸残基的序列。根据片段的排列，建立了酶的完整一级结构。氨基酸序列对应于65，424的多肽部分的分子量。包括己糖胺和中性碳水化合物含量，计算出本发明G1样品的分子量为约82，000。G1的大部分碳水化合物存在于70个氨基酸残基的高度糖基化区域中，该区域包含约35个O-糖基丝氨酸和苏氨酸残基。该区域终止于距离酶的C-末端约100个残基。在多肽链的中心部分发现两个N-糖基化位置。该分子含有9个半胱氨酸残基。糖淀粉酶序列与各种α-淀粉酶之间没有明显的同源性。
The primary structure of glucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of G1 were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517–527 (1983)), identification of 574 of the 614 amino acid residues in G1. Sequencing of glucoamylase G1 cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.The majority of the carbohydrate of G1 is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various α-amylases.