天门冬酰胺酶基因在黑曲霉中的同源表达

Homologous Expression of Asparaginase Gene in Aspergillus niger

DOI：10.3969/j.issn.1673-1689.2015.05.018

中文关键词: 天门冬酰胺酶 黑曲霉 基因置换 Berthelot显色反应

英文关键词: asparaginase,Aspergillus niger,gene replacement,berthelot colour reaction

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中文摘要:

根据NCBI中的黑曲霉天门冬酰胺酶基因asp序列(GI:145231287)设计特异性引物,从黑曲霉CICC2462基因组中扩增asp基因编码区,构建其黑曲霉表达载体pSZHG-Asp。通过农杆菌介导法转化黑曲霉,筛选以asp基因置换糖化酶基因glaA的同源重组转化子。对转化子的表达产物进行SDS-PAGE分析和酶活检测。获得8株在glaA位点发生基因置换的同源重组转化子,并对其中4株的上清液进行检测。SDS-PAGE结果显示,在42 000处有目的蛋白质条带,重组菌株的目的蛋白质表达量为185~417 μg/mL,发酵液最高酶活为21 111 U/mL。

英文摘要:

In order to achieve the homologous expression of asparaginase gene asp in Aspergillus niger CICC2462,the asparaginase engineering strains were construced. The specific primers were designed according to asparaginase gene asp sequence in the NCBI(GI:145231287)to amplify the coding region of asp and pSZHG-asp expression vector was constructed. Then agrobacterium mediated method was utilized to transform Aspergillus niger,and homologous recombinations which glucoamylase gene(glaA) was replaced by Asp were screened. The expression products of the transformant were analyzed using SDS-PAGE and enzyme activity detection. Eight homologous recombination strains,where glaA locus was replaced,were obtained,and the supernatant in 4 of them were measured. The result of SDS-PAGE showed protein bands at 42 000,the amount of the recombinated protein was 185~417 μg/mL,and the highest enzyme activity of fermentation broth was up to 21 111 U/mL.

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