Frederick K R, Tung J, Emerick R S, Masiarz F R, Chamberlain S H, Vasavada A, Rosenberg S, Chakraborty S, Schopfer L M
Chiron Research Labs, Chiron Corporation, Emeryville, California 94608.
J Biol Chem. 1990 Mar 5;265(7):3793-802.
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.
利用从黑曲霉葡萄糖氧化酶（EC 1.1.3.4）肽段氨基酸序列推导而来的寡核苷酸探针，已从cDNA文库和基因组文库中克隆出该酶的基因。成熟酶由583个氨基酸组成，前面有一个22个氨基酸的前导序列。在编码区内未发现间隔序列。该酶含有3个半胱氨酸残基和8个潜在的N-糖基化位点。该蛋白与多形汉逊酵母的醇氧化酶有26%的同源性，其N端有一段序列与其他黄素酶如对羟基苯甲酸羟化酶和谷胱甘肽还原酶的AMP结合区域同源。已构建了重组酵母表达质粒，其含有酵母乙醇脱氢酶II-3-磷酸甘油醛脱氢酶杂交启动子、酵母α-因子信息素前导序列或葡萄糖氧化酶前导序列以及成熟葡萄糖氧化酶编码序列。当这些质粒转化到酵母中时，它们指导合成并分泌75至400微克/毫升的活性葡萄糖氧化酶。对酵母来源的酶进行分析表明，它们具有相当的比活性，并且比黑曲霉蛋白具有更广泛的N-糖基化。
