嗜热真菌Thermomyces lanuginosus热稳定几丁质酶基因的cDNA克隆及表达

根据Thermomyces lanuginosus热稳定几丁质酶Chit的N-端氨基酸序列和同源保守序列设计简并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该几丁质酶的编码基因chit,全长cDNA为1500bp,包含一个由442个氨基酸组成的开放阅读框。该基因已在GenBank中注册,登录号为DQ092332。将成熟肽几丁质酶Chit阅读框与酵母表达载体pPIC9K连接,构建重组质粒pPIC9K/chit,转化毕赤酵母GS115,在甲醇的诱导下,成功地分泌出具生物活性的几丁质酶,诱导6d后酶活性达2.261U/mL,酶蛋白表达量为0.36mg/mL。该酶的最适反应温度和pH值分别为60℃和5.5,该酶在50℃以下稳定;65℃的半衰期为40min。

cDNA cloning and expression of thermostable chitinase from thermophilic fungus Thermomyces lanuginosus

A chitinase-encoding gene from Thermomyces lanuginosus was cloned by Rapid Amplification of cDNA Ends (RACE) using degenerated oligonucleotide primers designed from N-terminal amino acid sequence and the conserved amino acid sequence. The cloned full-length cDNA named chit, 1500bp in size, contained an OFR with 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acids sequence showed the catalytic domain of chit was high homology with the catalytic domains of the other chitinases in family-18, contained 2 conserved motifs related with catalytic activity of chitinase. The recombinant plasmid was generated by inserting the ORF of mature protein Chit into expression vector pPIC9K, and transformed into Pichia pastoris GS115. The recombinant chitinase was secreted successfully with the expression level of 0.36mg/mL and its activity could reach to 2.261U/mL after methanol induction 6d. The recombinant chitinase exhibited optimum catalytic activity at pH 5.5 and 60 degrees C. The enzyme was stable at 50 degrees C and the half life time at 65 degrees C was 40min.