篮状菌Talaromyces leycettanus JCM12802来源的高温葡萄糖淀粉酶性质与结构

Characterization and structure of a novel thermostable glucoamylase from Talaromyces leycettanus JCM12802

摘要:

葡萄糖淀粉酶作为淀粉糖化的关键用酶之一，广泛应用于食品、医药和发酵工业等行业。由于整个制糖过程都是在高温下完成的，因此对葡萄糖淀粉酶的反应温度和热稳定性有较高要求。本研究从嗜热篮状菌Talaromyces leycettanus JCM12802中克隆到一个糖苷水解酶第15家族 (GH15) 葡萄糖淀粉酶基因 (Tlga15A) 并在毕赤酵母GS115 中实现异源表达。重组葡萄糖淀粉酶TlGA的最适pH为4.5，在75 ℃下表现出最高酶活。TlGA热稳定性好，65 ℃条件下处理1 h剩余70%以上酶活力；70 ℃处理30 min后仍有43%酶活力。TlGA有较强的离子抗性和宽泛的底物特异性，TlGA水解可溶性淀粉、支链淀粉、糖原、糊精和普鲁兰的比活力分别为 (255.6± 15.3) U/mg、(342.3±24.7) U/mg、(185.4±12.5) U/mg、(423.3±29.3) U/mg和 (65.7±8.1) U/mg。从葡萄糖淀粉酶TlGA的一级结构、二级结构和三级结构3个层面对其进行比较分析，发现一级结构中较少的Gly组成和三级结构中较低的非极性基团溶剂可及表面积可能是维持葡萄糖淀粉酶TlGA温度稳定性的主要原因。综合其性质特点和对结构的分析，葡萄糖淀粉酶TlGA在工业葡萄糖生产中有较大应用潜力。

Abstract:

Glucoamylase is a critical ingredient for saccharification in the starch decomposition, and widely used in food, pharmaceutical and fermentation industries. Glucoamylases are usually thermostable and have peak activities at high temperature, as required for the industrial process of glucose production. In this study, a glucoamylase gene belonging to the glycoside hydrolase (GH) family 15, Tlga15A, was cloned from Talaromyces leycettanus JCM12802, and successfully expressed in Pichia pastoris GS115. Recombinant glucoamylase TlGA showed optimal activities at pH 4.5 and 75 °C. The result of thermostability analysis showed that TlGA retained above 70% activity after incubating for 1 h at 65 °C, and 43% residual activity after 30 min at 70 °C. Moreover, TlGA had high resistance to most metal ions and chemical reagents tested. Various starch substrates could be hydrolyzed by TlGA, including soluble starch (255.6±15.3) U/mg, amylopectin (342.3± 24.7) U/mg, glycogen (185.4±12.5) U/mg, dextrin (423.3±29.3) U/mg and pullulan (65.7±8.1) U/mg. The primary, secondary and tertiary structures of glucoamylase were further analyzed. The low ratio of Gly in the primary structure and low exposed nonpolarity solvent accessible surface in the tertiary structure may be the main reasons for TlGA’s thermostability. These results show that TlGA is great promising for potential use in the commercial production of glucose syrups. Moreover, this research will provide knowledge and innovating ideas for the improvement of glucoamylase thermostability.