重组嗜热乳糖酶在毕赤酵母中的表达、纯化与活性分析

doi:10.3969/j.issn.1673-1689.2018.08.005

首页 > 过刊浏览>2018年第37卷第8期 >812-816. DOI:10.3969/j.issn.1673-1689.2018.08.005

重组嗜热乳糖酶在毕赤酵母中的表达、纯化与活性分析
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                        10.3969/j.issn.1673-1689.2018.08.005
                    
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Expression，Purification and Activity Assay of Recombinant Thermophile Lactase From Pichia pastoris

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为获得耐高温的重组乳糖酶，PCR扩增激烈热球菌（Pyrococcus furious）乳糖酶基因、构建毕赤酵母分泌型表达载体pPICZαA/Lac并转化毕赤酵母X-33菌株。重组酵母转化子经甲醇诱导，重组嗜热乳糖酶实现了分泌表达并经蛋白印迹验证。纯化前上清液中乳糖酶总活力高达125 U/mL，经镍亲合纯化得重组酶，SDS-PAGE显示其纯度在95%以上，比活力1 800 U/mg，该酶最适温度在105 ℃左右且热稳定性好，具有很强的水解乳糖能力。

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In order to obtain the recombinant thermostable lactase，a DNA fragment containing the mature lactase gene was amplified from Pyrococcus furious by PCR and cloned into pPICZaA，generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant lactase production was identified by Western blotting，secreting as much as 125 U/mL induction by methanol. The recombinant thermostability lactase was purified by Ni+-NTA affinity chromatography and SDS-PAGE results shown that the purity was over 95%. The specific activity was about 1 800 U/mg and the optimal temperature was about 105 ℃. It was also showed that the purified lactase from P. pastoris has a highly thermostability and strong ability to hydrolysis lactose.

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李洪波,罗海燕,张树琴,吴东海.重组嗜热乳糖酶在毕赤酵母中的表达、纯化与活性分析[J].食品与生物技术学报,2018,37(8):812-816.

LI Hongbo, LUO Haiyan, ZHANG Shuqin, WU Donghai. Expression，Purification and Activity Assay of Recombinant Thermophile Lactase From Pichia pastoris[J]. Journal of Food Science and Biotechnology,2018,37(8):812-816.

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在线发布日期: 2018-09-29
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