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为研究黄蓝状菌(Talaromyces flavus)几丁质酶性质和作用,通过RT-PCR、3’RACE及5’TAIL-PCR技术克隆了一个黄蓝状菌几丁质酶基因tfchi1,该基因全长为2 561 bp,含有6个内含子,包含一个1 194 bp的ORF,编码397个氨基酸,分子量为43.47 kDa,属于糖苷水解酶第18家族。利用基因重组的方法构建酵母分泌型表达载体pPIC9K/tfchi1,并转化毕赤酵母,筛选得到一株高效表达几丁质酶的工程菌GS-TFCHI1-9,甲醇诱导第7 d酶活性最高,达32.29 U.mL-1。SDS-PAGE检测纯化重组蛋白的分子量约44 kDa。重组几丁质酶Tfchi1最适反应温度为35℃,最适反应pH值为5.5,在pH 6～8之间稳定。Zn2+、Cu2+对Tfchi1活性有明显的抑制作用。抑菌活性测定结果显示,Tfchi1可明显抑制敏感植物病原真菌的菌丝生长和分生孢子萌发。该酶在几丁质资源的开发利用和生物防治等领域具有较广的应用前景。
In order to study the properties and effects of chitinase from Talaromyces flavus, a chlamydophila chitinase gene tfchi1 was cloned by RT-PCR, 3’RACE and 5’TAIL-PCR. It is 2 561 bp in length and contains 6 introns. It contains a 1 194 bp ORF encoding 397 amino acids with a molecular mass of 43.47 kDa and belongs to the 18th family of glycoside hydrolases. The yeast secreting expression vector pPIC9K / tfchi1 was constructed by gene recombination and transformed into Pichia pastoris. The results showed that GS-TFCHI1-9, an engineering bacterium with high expression of chitinase, 32.29 U.mL-1. The molecular weight of purified recombinant protein was about 44 kDa by SDS-PAGE. The optimal reaction temperature of recombinant chitinase Tfchi1 was 35 ℃, and the optimum reaction pH was 5.5, which was stable at pH6 ~ 8. Zn2 +, Cu2 + on Tfchi1 activity was significantly inhibited. The results of antibacterial activity assay showed that Tfchi1 could significantly inhibit mycelial growth and conidial germination of susceptible plant pathogenic fungi. The enzyme has a wide range of applications in the fields of development and utilization of chitin resources and biological control.