球毛壳菌α-葡聚糖酶的异源表达、纯化及特性表征

doi:10.3969/j.issn.1673-1689.2021.11.004

首页 > 过刊浏览>2021年第40卷第11期 >30-38. DOI:10.3969/j.issn.1673-1689.2021.11.004

球毛壳菌α-葡聚糖酶的异源表达、纯化及特性表征
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                        10.3969/j.issn.1673-1689.2021.11.004
                    
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Heterologous Expression, Purification and Characterization of Alpha-Glucanase from Chaetomium globosum

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利用毕赤酵母密码子偏好性优化合成球毛壳菌α-葡聚糖酶基因，并在毕赤酵母GS115中实现异源表达。通过高拷贝筛选和摇瓶发酵条件的单因素优化，重组α-葡聚糖酶的酶活力由初始的2.692 U/mL提高至47.915 U/mL。选择 Hitrap Q HP 和Hitrap SP HP 的双步层析分离将发酵粗酶液纯化至电泳纯，纯化倍数40.83，比活达277.61 U/mg，回收率为24.15%。纯酶最适温度和pH分别为60 ℃和5.5。在20~50 ℃和pH为4.5~8.5范围内稳定性良好，浓度为10 mmol/L的Fe²⁺对酶有激活作用，Cu²⁺浓度（0.1~10 mmol/L）越高对酶的抑制作用越强。酶动力学实验发现该重组酶对高相对分子质量的底物亲和力更高，葡聚糖T2000为该酶的最适底物。

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In this study, α-glucanase derived from Chaetomium globosum was synthesized and optimized according to the codon preference of <>Pichia pastoris, and then successfully heterologous expressed in Pichia pastoris GS115. Through the high-copy screening and single-factor optimization of shaike flask fermentation, the enzyme activity of recombinant α-glucanase was increased from 2.692 U/mL to 47.915 U/mL. The crude enzyme was purified to electrophoretic purity by two-step chromatography through Hitrap Q HP and Hitrap SP HP. The purification factor reached 40.83, with the recovery of 24.15%. And the specific activity of purified enzyme reached 277.61 U/mg. The optimal reaction temperature and pH were 60 ℃ and 5.5, respectively. The enzymatic activity kept stable within 20~50 ℃ and pH 4.5~8.5. The presence of 10 mM Fe²⁺ activated the enzyme, whereas 0.1~10 mM Cu²⁺ inhibited the enzyme. The higher concentration of Cu²⁺, the stronger inhibition of enzyme. Affinity study indicated that the recombinant enzyme had the higher affinity towards substrates with higher molecular weight. Dextran T2000 was the optimal substrate of the enzyme.

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顾莉莉,周楠迪,田亚平.球毛壳菌α-葡聚糖酶的异源表达、纯化及特性表征[J].食品与生物技术学报,2021,40(11):30-38.

GU Lili, ZHOU Nandi, TIAN Yaping. Heterologous Expression, Purification and Characterization of Alpha-Glucanase from Chaetomium globosum[J]. Journal of Food Science and Biotechnology,2021,40(11):30-38.

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在线发布日期: 2021-11-30
出版日期: 2021-11-25

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