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首页> 外文期刊>Yeast >An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme
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An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme

机译：来自双形酵母腺病毒的胞外脂肪酶：ALIP1基因的分子克隆和纯化的重组酶的表征

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摘要

The lipase-encoding Arxula adeninivorans ALIP1 gene was isolated using fragments of lipase isolates obtained by trypsin digestion for the definition of oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1347 bp encoding a 420 amino acid protein of some 50 kDa preceded by an N-terminal 28 prepro-secretion sequence. The deduced amino acid sequence was found to be similar to the lipases from Candida albicans and C. parapsilosis (34-38% identity) and more distantly related to other lipases. The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases. The expression of the gene is regulated by carbon source. In media supplemented with Tween 20, induction of the ALIP1 gene and accumulation of the encoded lipase in the medium is observed, thus demonstrating gene regulation by lipophilic compounds. The enzyme characteristics are analysed from isolates of native strains as well as from those of recombinant strains expressing the ALIP1 gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins a molecular mass of 100 kDa was determined, indicating a dimeric structure, a pH optimum at pH 7.5 and a temperature optimum at 30 degrees C. The enzyme hydrolyses all ester bonds in all triglyceride substrates tested. Middle-sized chain fatty acids are more efficiently hydrolysed than short- and long-chain fatty acids, with the highest activity on C8/C10 fatty acid esters pNP-caprylate, pNP-caprate and tricaprylin.
机译：使用通过胰蛋白酶消化获得的脂肪酶分离物的片段，通过PCR筛选方法定义寡核苷酸引物，分离出编码脂肪酶的Arxula adeninivorans ALIP1基因。该基因包含一个1347 bp的ORF，编码约50 kDa的420个氨基酸蛋白，并带有一个N端28个预分泌前序列。发现推导的氨基酸序列与来自白色念珠菌和副念珠菌的脂肪酶相似（同一性为34-38％），并且与其他脂肪酶的亲缘关系更远。该序列包含共有五肽基序（-Gly-X-Ser-X-Gly-），该基序形成脂肪酶中界面脂质识别位点的一部分。基因的表达受碳源调节。在添加了Tween 20的培养基中，观察到ALIP1基因的诱导和培养基中编码脂肪酶的积累，从而证明了亲脂性化合物对基因的调控。从天然菌株的分离株以及表达ALIP1基因的重组菌株的分离株中分析酶的特性，所述菌株在强力的腺嘌呤衍生自TEF1启动子的控制下。对于这两种蛋白质，测定的分子量均为100 kDa，表明其为二聚体结构，最适pH值为7.5，最适温度为30℃。该酶水解了所有测试的甘油三酯底物中的所有酯键。中链脂肪酸比短链和长链脂肪酸更有效地水解，对C8 / C10脂肪酸酯pNP-辛酸酯，pNP-癸酸酯和三辛酸酯具有最高的活性。

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