植酸酶产生菌黑曲霉N14的诱变选育及其基因分析

Selection of Aspergillus niger14 Strain Producing Phytase and Sequence Analysis of its Phytase phyA Gene

以植酸酶产生菌黑曲霉03214为出发菌株,经紫外线和亚硝基胍诱变,获得了产酶活性较出发菌株提高了22.3%,达422IU/ml发酵液的突变菌株黑曲霉N14,其最适pH值为2.5,最适温度为50℃。通过对黑曲霉N14植酸酶phyA基因进行PCR扩增,获得了一条长约1.5kb的特异性产物。以pMD18 T为载体,构建了含有目的基因片段的重组质粒。DNA序列测定表明,目的基因片段含有植酸酶phyA基因的完整序列(GenBankAccession:AY426977),phyA基因全长1506bp,其中包含一段长102bp的内含子,编码467个氨基酸,有10个潜在的糖基化位点,5'端有一编码19个氨基酸的信号肽序列。实验结果为植酸酶基因工程菌的构建奠定了基础。

<Abstrcat> A mutant strain Aspergillus niger 14 with higher phytase activity than its parental strain Aspergillus niger 03214 was selected by treatment with ultraviolet and N-methyl-N-nitro-N-nitrosoguanidine. The optimal pH and temperature of the activity of the phytase produced by Aspergillus niger 14 were 2.5 and 50 ℃ respectively. Cultivated by a shaking method, the phytase of the mutant strain was produced 422 U/ml. The phyA gene encoding phytase was cloned from Aspergillus niger 14 by the polymerase chain reaction (PCR). Nucleotide sequence analysis of the phyA gene revealed that the coding region is 1506 bp in size (Accession: AY426977), including a 102 bp intron, and encoding a peptide of 467 amino acid residues, in which there is a signal peptide of 19 amino acids and a mature peptide of 448 amino acids. There are ten potential N-glycosylation sites in phytase. Comparison with the phyA gene from A.niger NRRL3135 shows 96% homology in nucleotides and 98% homology in amino acid residues.