β-甘露聚糖酶AuMan5A^loop/H321G在乳酸克鲁维酵母中的高效表达

High-Level Expression of A β-Mannanase AuMan5A^loop/H321Gin Kluyveromyces lactis GG799

DOI：10.3969/j.issn.1673-1689.2019.07.004

中文关键词: β-甘露聚糖酶 乳酸克鲁维酵母 表达 诱导剂 发酵工艺

英文关键词: β-mannanase,Kluyveromyces lactis,expression,inducer,fermentation process

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中文摘要:

为制备食品级β-甘露聚糖酶，将来源于宇佐美曲霉（Aspergillus usamii） 的糖苷水解酶5家族β-甘露聚糖酶突变体基因（Auman5A^loop/H321G） 克隆至表达质粒pKLAC1信号肽?琢-MF下游，构建重组表达质粒pKLAC1-Auman5A^loop/H321G，并电转化乳酸克鲁维酵母（） GG799。采用半乳糖诱导表达和酶活性分析获得了高水平表达的重组β-甘露聚糖酶突变酶（reAuMan5A^loop/H321G） 的转化子GG799/Auman5A^loop/H321G，其产酶活力为54.9 U/mL。SDS-PAGE分析显示，reAuMan5A^loop/H321G的表观相对分子质量约为55 000。经YPG培养基初始pH、摇瓶装液量以及诱导剂的种类、初始质量浓度和添加方式的初步优化，GG799/Auman5A^loop/H321G所产酶活力较工艺优化前提高了254.6%，从而实现了reAuMan5A^loop/H321G在乳酸克鲁维酵母中的高效表达。

英文摘要:

To prepare the food-grade β-mannanase，a β-mannanase mutant geneAuman5A^loop/H321Gwas expressed in Kluyveromyces lactis GG799.Auman5A^loop/H321G，gene constructed based on a glycoside hydrolase family 5 β-mannanase gene（Auman5A） from Aspergillus usamii，was inserted into the signal peptide α-MF downstream of the expression plasmid pKLAC1，forming a recombinant plasmid pKLAC1-Auman5A^loop/H321G. Then，pKLAC1-Auman5A^loop/H321Gwas electroporated into Kluyveromyces lacti GG799. After induction by galactose and enzyme activity analysis，a transformant GG799/Auman5Aloop/H321G expressing the highest recombinant β-mannanase（reAuMan5A^loop/H321G） activity of 54.9 U/mL was selected for subsequent studies. The apparent molecular weight of reAuMan5A^loop/H321Gwas estimated to be about 55 000 by SDS-PAGE analysis. Furthermore，the β-mannanase activity produced by GG799/Auman5A^loop/H321Gwas increased by 254.6% after primary optimization of the initial pH and loading volume of YPG medium as well as the species，initial concentration and supplementary mode of inducer. As a result，the high-level expression of reAuMan5A^loop/H321GinK. lactiswas actualized at the optimized fermentation process.

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