嗜热毛壳菌纤维素酶(CBHⅡ)cDNA的克隆及在毕赤酵母中的表达

Cloning and Expressing of Cellulase Gene(cbh2) from Thermophilic Fungi Chaetomium thermophilum CT2

嗜热毛壳菌Chaetomium thermophilumCT2是一种土壤腐生菌,可产生具有重要工业生产价值的纤维素酶类。RACEPCR获得嗜热毛壳菌纤维二糖水解酶Ⅱ(CBHⅡ)的编码基因(cbh2)。DNA序列分析表明cbh2的开放阅读框由1428个碱基组成,编码476个氨基酸。推断的氨基酸序列包含一个典型真菌纤维素酶的糖结合域(CBD)、催化域(CD)以及二者之间富含脯氨酸和羟基氨基酸的连接桥。根据氨基酸序列推算该酶分子量为53kD,属于糖苷水解酶第六家族,具有该家族催化保守区的典型特征。PCR扩增cbh2的成熟蛋白编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶蛋白的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.2mgmL。经硫酸铵沉淀、DEAE-Sepharose Fastflow阴离子层析等步骤纯化了该重组表达蛋白。SDSPAGE得到重组蛋白分子量为67kD,与从嗜热毛壳菌中纯化的该酶分子量一致。该重组纤维二糖水解酶作用的最适合温度50℃,最适pH4.0,在70℃的半衰期为30min,具有较好的热稳定性。

Chaetomium thermophilum CT2 can produce extracellular cellulase with industrial value. We designed two degenerate primers to amplify catalytic domain sequence of cellobiohydrolase Ⅱ （ CBH Ⅱ ）. Full length of cDNA was obtained by rapid amplification of cDNA ends technologies. DNA sequencing revealed that cbh2 has an open reading frame of 1428bp, which encodes a putative polypeptide of 476 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 53 kD and the cbh2 consists of a fungal-type carbohydrate binding domain （CBD） separated from a catalytic domain by a linker region rich in proline/serine/threonine. PCR product consisting of the entire CBH Ⅱ coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. Highly efficient production of the cellobiohydrolase Ⅱ was achieved in P. pastoris under the control of the AOX1 promoter, and the expressing level was 1.2 mg/mL by small-scale culturing. The recombinant cellobiohydrolase Ⅱ was purified by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography. A molecular mass of the purified enzyme is 67 kD determined by SDSPAGE and this is similar to the native cellobiohydrolase Ⅱ purified from C. thermophilum CT2. The recombinant enzyme exhibited optimum catalytic activity at pH 4.0 and 50℃ respectively. It was thermostable at 50℃ and retained 50% of its original activity after 30 min at 70℃ . The high level of fully active recombinant cellobiohydrolase Ⅱ got from P. pastoris makes this expression system attractive for fermentor and industrial applications.