Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae
Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae
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DOI：
10.1111/j.1574-6968.1996.tb08462.x
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发表时间：
1996-09-15
影响因子：
2.1
通讯作者：
Sekiguchi, J
中科院分区：
文献类型：
--
作者：
Tsuchiya, A;Nakazawa, H;Sekiguchi, J
米曲霉IFO4202至少产生两种胞外脂解酶L1和L2（分别为角质酶以及单酰甘油和二酰甘油脂肪酶）。用根据L2的氨基酸序列合成的23聚寡核苷酸作为探针，对限制性内切酶消化的基因组DNA片段进行Southern杂交，结果表明存在L2基因（暂定为mdlB）以及一个额外的弱杂交区域。一个含有基因组mdlB基因的片段在大肠杆菌中被克隆。对该片段进行核苷酸测序，发现一个开放阅读框，由1021个核苷酸组成，其中包含两个内含子（分别为51和52个核苷酸）。在终止密码子下游182和287bp处发现了假定的多聚腺苷酸化信号。mdlB基因推导的氨基酸序列对应306个氨基酸残基，包括一个28个氨基酸的前导序列，并且与卡门培尔青霉的mdlA基因高度相似。被认为构成脂肪酶催化三联体（丝氨酸、天冬氨酸和组氨酸）的三个残基也保守。
Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively). Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23-mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB) and an additional weakly hybridizing region. A fragment containing the genomic mdlB gene was cloned in Escherichia coli. Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides). Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon. The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii. Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.