Guo Run-fang, Li Duo-Chuan
Department of Environmental Biology, Shandong Agricultwural University, Tai' an 271018, China.
Wei Sheng Wu Xue Bao. 2006 Feb;46(1):99-103.
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.
利用根据N端氨基酸序列和保守氨基酸序列设计的简并寡核苷酸引物，通过cDNA末端快速扩增（RACE）技术，从嗜热栖热菌中克隆了一个几丁质酶编码基因。克隆得到的全长cDNA名为chit，大小为1500bp，包含一个编码442个氨基酸的开放阅读框（OFR）。该基因chit已在GenBank中注册，登录号为DQ092332。推测氨基酸序列的比对结果表明，chit的催化结构域与18家族中其他几丁质酶的催化结构域具有高度同源性，包含2个与几丁质酶催化活性相关的保守基序。通过将成熟蛋白Chit的开放阅读框插入表达载体pPIC9K中构建重组质粒，并将其转化到毕赤酵母GS115中。重组几丁质酶成功分泌，甲醇诱导6天后表达水平为0.36mg/mL，活性可达2.261U/mL。重组几丁质酶在pH 5.5和60℃时表现出最佳催化活性。该酶在50℃时稳定，在65℃时的半衰期为40分钟。
