嗜热裂孢菌木聚糖酶在毕赤酵母中的表达及酶学性质

doi:10.3969/j.issn.1673-1689.2016.05.007

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嗜热裂孢菌木聚糖酶在毕赤酵母中的表达及酶学性质
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                        10.3969/j.issn.1673-1689.2016.05.007
                    
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Expression of A Xylanase from Thermobifide fusca in Pichia pastoris And Its Characterization Analysis

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摘要:

将优化后的来自嗜热裂孢菌的木聚糖酶基因在毕赤酵母中进行异源表达并分析了其表达产物的酶学性质。对来源于嗜热裂孢菌的木聚糖酶基因催化域进行密码子优化，人工合成优化后的木聚糖酶成熟肽基因xyl11M，构建重组表达质粒pPIC9K-xyl11M，将其经SalⅠ线性化后电击转化毕赤酵母GS115，经G418筛选得到重组工程菌GS115/xyl11M，甲醇诱导表达重组蛋白。表达的重组蛋白reXyl11M经SDS-PAGE分析，其相对分子质量约34 000，酶活性可达到105.3 U/mL，最适反应温度为70 ℃，并在30~75 ℃温度内较稳定；最适反应pH为6.0，在pH 6.5~7.5范围内稳定；Co2+、Ba2+、Cu2+、Li+对酶活性有强烈的激活作用，Fe3+有明显的抑制作用，其它金属离子及EDTA对reXyl11M酶活性影响不大。结果表明xyl11M成功在毕赤酵母GS115中实现表达，且reXyl11M的酶学特性优良。

Abstract:

A codon-optimized mature peptide gene encoding a thermostable xylanase from Thermobifide fusca has been cloned into the expression plasmid pPIC9K and named as pPIC9K-xyl11M. The pPIC9K-xyl11M was linearized with SalⅠ and integrated into the genome of Pichia pastoris GS115 by electroporation. The recombinant P. pastoris GS115/xyl11M was screened by G418 and then was induced with methanol to express reXyl11M. The reXyl11M activity expressed by P. pastoris transformant reached 105.3 U/mL. The molecular weight of reXyl11M was estimated to be 34 000 by SDS-PAGE. The reXyl11M displayed the highest activity at 70 ℃ and pH 6.0. It was stable at a temperature range of 30~75 ℃，and at a pH range of 6.5~7.5. Co2+，Ba2+，Cu2+，Li+ had a strong enzyme activation effect，Fe3+ had obvious inhibitory effect，its activity was not significantly affected by other metal ions and EDTA. This revealed that reXyl11M was successfully expressed in P. pastoris and had good enzymatic characterizations.

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卢桂义,邬敏辰,黄卫宁,张峦,李宁,Courtin Christopher.嗜热裂孢菌木聚糖酶在毕赤酵母中的表达及酶学性质[J].食品与生物技术学报,2016,35(5):492-497.

LU Guiyi, WU Minchen, HUANG Weining, ZHANG Luan, LI Ning, Courtin Christopher. Expression of A Xylanase from Thermobifide fusca in Pichia pastoris And Its Characterization Analysis[J]. Journal of Food Science and Biotechnology,2016,35(5):492-497.

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在线发布日期: 2016-11-01
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