地衣芽孢杆菌YP1A耐有机溶剂蛋白酶基因的克隆与功能表达

根据B．licheniformis YP1A来源的碱性蛋白酶具有的高强度耐有机溶剂性能及相关数据库分析，采用PCR克隆B．licheniformis YP1A耐有机溶剂碱性蛋白酶基因，序列分析显示该基因（1264bp）包含启动子与编码380个氨基酸的开放阅读框（ORF），ORF包括信号肽、前肽及编码254个氨基酸的成熟肽序列。相关基因分析表明，YP1A耐有机溶剂碱性蛋白酶基因与地衣芽孢杆菌ATCC14580的碱性蛋白酶基因仅有6个氨基酸残基差异：构建2种含YP1A碱性蛋白酶CDS的组成型穿梭表达载体pHY／aprYP与pHY／aprP43，前者采用YP1A蛋白酶自带的启动子，后者则采用来自于质粒pP43NMK的P43强启动子。利用这2种表达载体在枯草芽孢杆菌WB800中成功进行蛋白酶的功能表达．其中P43强启动子的表达能力明显优于碱性蛋白酶自带的启动子，表达的蛋白酶比酶活为395U／ml。重组菌表达的碱性蛋白酶在体积分数50％的亲水及疏水有机溶剂中表现出了很好的耐受性，验证了克隆基因为地衣芽孢杆菌YP1A的高强度耐有机溶剂碱性蛋白酶基因.

Cloning and functional expression of organic-solvent tolerant protease from B.licheniformis YP1A

Alkaline protease secreted by B. licheniformis YP1A showed high tolerance characteristics to organic solvents. According to the properties of the protease and the analysis of related genes of typical strain ATCC14580 in GeneBank, the alkaline protease (APR) was cloned and its nucleotides were sequenced. Sequence analysis revealed that the APR gene composed of 1 264 bp that encoded its promoter and an ORF of 380 amino acid residues. The ORF contained signal peptide, precursor and mature peptide of 254 amino acids. There are 6 mutations of amino acid residues between aprYP and kerA from B.licheniformis ATCC14580. The plasmids pHY/aprYP and pHY/aprP43 carrying aprYP with aprYP upstream promoter and with P43 promoter from the vector of pP43NMK were constructed and transduced into B.subtilis WB800, respectively. The active expression of aprYP in these two recombinants was obtained. The recombinant WB800-pHY/aprP43 with P43 promoter showed a relative efficient expression and protease activity reached 395 U/mL. The protease expressed by the recombinant encoding aprYP was remarkably tolerant in some hydrophobic and hydrophilic organic solvents at concentration of 50% (V/V). The result that the gene of APR encoded the previous reported solvent-stable alkaline protease of B.licheniformis YP1A.