草酸青霉外切葡聚糖酶CBHⅠ基因的克隆

Cloning of an Exo-β-1,4-D-glucanases CBHⅠGene from Penicillium oxalicum

以草酸青霉SJ1菌株的基因组DNA和总RNA,利用PCR和RT-PCR技术进行扩增得到外切葡聚糖酶CBHⅠ(ExocellobiohydrolaseⅠ)基因的序列.CBHⅠ基因DNA序列全长1 638 bp,无内含子,编码545个氨基酸的多肽,推测其蛋白分子大小约为57 ku,蛋白的p I值为4.90.预测结果表明,CBHⅠ是一种很强疏水性的蛋白,具有43个磷酸化修饰位点,蛋白质三级结构以β-折叠为主,根据蛋白质序列比对分析,推测E236、D238和E241这3个氨基酸为酶的活性位点,为后续利用分子改造方法提高青霉产纤维素酶的能力提供理论依据.

By extracting genome DNA and total RNA of Penicillium oxalicum SJ1 strains, the sequence of exo-β-1 ,4-D-glucanases CBH I gene was cloned by PCR and RT-PCR. The total length of CBH I gene was 1 683 bp without introns, encoding 545 amino acids. Theoretical isoelectric point of CBH I protein was 4. 90. Relative molecular mass was 53 ku. Bioinformatics predic- tion revealed that CBH I was a kind of very strong hydrophobic proteins, containing 43 phosphorylated modification sites, Protein tertiary structure was given priority to with beta folding. According to the analysis of protein sequence alignment, three amino acids of speculation E236, D238 and E241 were enzyme＇s active sites, has laid a foundation for the subsequent study in the availability of high-performance engineering strains.