黑曲霉脂肪酶基因的克隆及其在毕赤酵母中的表达

根据黑曲霉F044脂肪酶N-端氨基酸序列，运用生物信息学方法，找到与黑曲霉脂肪酶基因同源的候选基因A84689．根据该基因序列，设计引物直接经RT—PCR扩增得到黑曲霉脂肪酶全长基因anl．anl全长1044bp，含3个内含子，编码297个氨基酸（含信号肽27个氨基酸），与其他脂肪酶基因没有明显的同源性．将成熟脂肪酶基因连接到pPIC9K载体上得到重组表达质粒，转化P．pastoris GSll5．脂肪酶基因在P．pastoris GSll5中实现了分泌性表达．

Cloning of the Lipase Gene of Aspergillus niger F044 and Its Expression in Pichia pastoris

From the N-terminal amino acid sequence of the Aspergillus niger F044 lipase,a potential homologous gene A84689 of the anl(the gene encoding the Aspergillus niger lipase) was found by means of bioinformatics.Based on the nucleotide sequence of the A84689,primers were designed to amplify anl.Nucleotide sequencing of the genomic DNA and cDNA revealed that the anl had an open reading frame comprising 1044 nucleotides,containing three introns of 54,45 and 51 bp.The deduced amino acid sequence of the anl encoded 297 amino acid residues including a signal sequence of 27 amino acid residues.The cDNA fragment coding the mature ANL(the protein of the Aspergillus niger lipase) was subcloned into the pPIC9K expression vector and the reconstructed expression plasmid pPIC9K-anl was then transformed into Pichia pastoris GS115.The anl was actively expressed in the Pichia pastoris GS115 and the recombinated lipase protein was secreted into the cultural broth.