| 引用本文： |
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朱婧,刘海余,王青艳,米慧芝,朱绮霞,廖思明,秦艳,申乃坤,黄日波.生淀粉结合域SBD及糖化酶基因在毕赤酵母中的融合表达[J].广西科学,2016,23(1):7-11. [点击复制]
- ZHU Jing,LIU Haiyu,WANG Qingyan,MI Huizhi,Zhu Qixia,LIAO Siming,QIN Yan,SHEN Naikun,HUANG Ribo.Fusion Expression of Raw Starch Binding Domain with Glucoamylase in Pichia pastoris[J].Guangxi Sciences,2016,23(1):7-11. [点击复制]
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| 摘要: |
| [目的]研究生淀粉结合域SBD及糖化酶基因在毕赤酵母中的融合表达,提高酶的表达量和水解生淀粉的能力。[方法]利用In-fusionTM PCR克隆技术将淀粉结合域SBD无缝隙插入到黑曲霉糖化酶基因glu的5'端构建融合表达质粒pPIC9K-psg,实现融合酶基因psg在毕赤酵母GS115中的高效表达,并进行酶学性质研究。[结果]融合酶的最适作用条件及热稳定性均与原始酶无明显差别,但反应温度及pH值的范围更为宽泛,在反应温度60~70℃,pH值为4.0~7.0时均较稳定;融合酶PSG降解生淀粉的能力较原始酶PG提高29.6%,比原始菌株提高86.5%。[结论]淀粉结合域SBD的融合提高了糖化酶水解生淀粉的能力。 |
| 关键词: 黑曲霉ASP-S21 生淀粉糖化酶 淀粉结合域 In-fusion TM PCR Cloning 融合表达 毕赤酵母GS115 |
| DOI：10.13656/j.cnki.gxkx.20160315.018 |
| 投稿时间：2015-01-27修订日期：2015-03-12 |
| 基金项目:国家自然科学基金项目(No.31160023),广西科学研究与技术开发计划项目(桂科合14123001-19),广西自然科学基金项目(No.2013GXNSFBA019102),八桂学者建设工程专项经费和2014年留学人员科技活动项目资助。 |
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| Fusion Expression of Raw Starch Binding Domain with Glucoamylase in Pichia pastoris |
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ZHU Jing, LIU Haiyu, WANG Qingyan, MI Huizhi, Zhu Qixia, LIAO Siming, QIN Yan, SHEN Naikun, HUANG Ribo
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| (National Engineering Research Center for Non-food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China) |
| Abstract: |
| [Objective] In order to improve the expression of glucoamylase fromAspergillus niger ASP-S21 and the hydrolysis ability of raw starch, the fusion expression of starch binding domain (SBD)with glu was conducted in Pichia pastoris GS115.[Methods] The fusion expression plasmid was constructed by inserting the SBD fragment to 5'-end of glucoamylase gene glu with In-fusionTM PCR Cloning technology.The recombinant enzyme (PSG)was over expressed in Pichia pastoris GS115 and its enzymatic properties were characterized.[Results] The PSG showed strong raw cassava starch hydrolyzing activity, which was 29.6% higher than the wildtype PG, and 86.5% higher than parental strain.The reaction temperature and pH of the acfusion enzyme were more broad than that of the wildtype.It was stable in temperature range of 60~70℃ and pH range from 4.0to 7.0.[Conclusion] The fusion of SBD with glucoamylase enables the fusion enzymes to hydrolyze raw starch more effectively. |
| Key words: Aspergillus niger ASP-S21 raw starch-digesting glucoamylase starch-binding domain (SBD) In-fusionTM PCR Cloning fusion expression Pichia pastoris GS115 |
