Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins
Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins
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DOI：
10.1385/mb:32:1:073
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发表时间：
2006-01-01
影响因子：
2.6
通讯作者：
Tan, TW
中科院分区：
文献类型：
--
作者：
Niu, WN;Li, ZP;Tan, TW
根霉菌的脂肪酶。对三酰甘油有很高的1，3-区域专一性，这使它们成为脂类修饰的重要酶。本研究利用巴斯德毕赤酵母成熟区基因(MRal)和含有前体序列的mRa基因，进行了重组根霉脂肪酶的胞外活性生产。分别筛选出两个含有mrA和prorA基因多拷贝的巴斯德毕赤酵母转化克隆，用于生产重组酶。在补料分批培养中，甲醇加入量由在线甲醇分析仪控制，培养92h上清液中含有91 mg/L重组前体脂肪酶和80 mg/L重组成熟脂肪酶。经超滤、SPSepharoseFast Flow层析和Butyl-SepharoseFast Flow层析纯化蛋白和蛋白。重组蛋白和重组蛋白的相对分子质量分别为32 kDa和29 kDa。氨基酸末端分析表明，该32 kDa蛋白与原序列羧基末端的28个氨基酸相连(RPro28rA)。与原序列羧基末端28个氨基酸结合的mRal和rRal的比脂肪酶活性分别为1543U/mg和2437U/mg。在pH 4.0-7.0范围内，rPro28的稳定性较高，而在pH 7.0-10.0范围内，rRal的稳定性较差。RPro28对三丁酸甘油酯(C-4)的脂肪酶活性最高，而rra对三辛胺(C-8)的脂肪酶活性最高。
The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizus lipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SPSepharose Fast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0-7.0, whereas rRAL was more stable at pH 7.0-10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C-4), whereas rRAL had the highest lipase activity toward tricaprylin (C-8).