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首页> 外文期刊>Protein Expression and Purification >Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp (BCC4124) in Pichia pastoris
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Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp (BCC4124) in Pichia pastoris

机译：毕赤酵母内生真菌Fusicoccum sp（BCC4124）的纤维二糖水解酶（CBHI）基因的鉴定与表达

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A gene encoding a cellobiohydrolase (CBHI) was isolated from Fusicoccum sp. (BCC4124), an endophytic fungus belongs in phylum Ascomycota, using 5' and 3' rapid amplification of cDNA end (RACE) technique. This CBHI gene contains 1395 nucleotides and encodes a 465-amino acid protein with a molecular weight of approximately 50 kDa. The deduced amino acid sequence showed significant similarity to those of other fungal CBHI belonging to family 7 of glycosyl hydrolase. Interestingly, the result from the amino acid alignment revealed that this CBHI does not contain the cellulose binding domain nor the linker region. The CBHI gene was successfully expressed in Pichia pastoris KM71. The purified recombinant CBHI has ability to hydrolyze Avicel, filter paper and 4-methylumbelliferyl P-D-cellobioside (MUC) but not carboxymethylcellulose (CMC). It showed an optimal working condition at 40 degrees C, pH 5 with K, and V-max toward MUC of 0.57 mM and 3.086 nmol/min/mg protein, respectively. The purified enzyme was stable at pH range of 3-11. The enzyme retained approximately 50% of its maximal activity after incubating at 70-90 degrees C for 30 min. Due to its stability through wide range of pH, and moderately stable at high temperature, this enzyme has potential in various biotechnology applications. (c) 2007 Elsevier Inc. All rights reserved.
机译：从Fusicoccum sp。分离到一个编码纤维二糖水解酶（CBHI）的基因。 （BCC4124）是一种内生真菌，属于子囊菌门，使用cDNA末端的5'和3'快速扩增（RACE）技术。该CBHI基因包含1395个核苷酸，并编码分子量约为50 kDa的465个氨基酸的蛋白质。推导的氨基酸序列显示出与糖基水解酶家族7的其他真菌CBHI的氨基酸序列显着相似。有趣的是，氨基酸比对的结果表明该CBHI不包含纤维素结合域也不包含接头区域。 CBHI基因在毕赤酵母KM71中成功表达。纯化的重组CBHI具有水解Avicel，滤纸和4-甲基伞形基P-D-纤维二糖苷（MUC）的能力，但不能水解羧甲基纤维素（CMC）的能力。它显示了在40摄氏度，pH为5，钾离子和V-max对MUC分别为0.57 mM和3.086 nmol / min / mg蛋白时的最佳工作条件。纯化的酶在3-11的pH范围内是稳定的。在70-90摄氏度下孵育30分钟后，该酶保留了其最大活性的约50％。由于其在广泛的pH范围内具有稳定性，并且在高温下具有中等稳定性，因此该酶在各种生物技术应用中具有潜力。 （c）2007 Elsevier Inc.保留所有权利。

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