Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme.
Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme.
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DOI：
10.1016/s0021-9258(19)39664-4
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发表时间：
1990-03
期刊：
影响因子：
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通讯作者：
K. Frederick;J. Tung;R. Emerick;F. R. Masiarz;S H Chamberlain;A. Vasavada;S. Rosenberg;S. Chakraborty;L. Schopfer;L. M. Schopter
中科院分区：
文献类型：
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作者：
K. Frederick;J. Tung;R. Emerick;F. R. Masiarz;S H Chamberlain;A. Vasavada;S. Rosenberg;S. Chakraborty;L. Schopfer;L. M. Schopter
黑曲霉葡萄糖氧化酶(EC 1.1.3.4)基因已从基因文库和基因组文库中克隆出来，该基因的氨基酸序列来源于黑曲霉葡萄糖氧化酶的多肽片段。成熟的酶由583个氨基酸组成，前面有22个氨基酸的前序。在编码区内未发现插入序列。该酶含有3个半胱氨酸残基和8个潜在的N-糖基化位点。该蛋白与Hansenuela Polya的乙醇氧化酶有26%的同源性，N端与对羟基苯甲酸羟基酶和谷胱甘肽还原酶等其他黄酮类酶的AMP结合区具有同源性。构建了酵母酒精脱氢酶II-甘油醛-3-磷酸脱氢酶复合启动子、酵母α因子信息素前导序列或葡萄糖氧化酶前序列以及葡萄糖氧化酶成熟编码序列的酵母表达载体。当转化到酵母中时，这些质粒会直接合成和分泌75到400微克/毫升的活性葡萄糖氧化酶。对酵母酶的分析表明，它们具有与黑曲霉蛋白相当的比活性，并且具有更广泛的N-连接糖基化作用。
The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.