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Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones.
Protein Expression and Purification ( IF 1.6 ) Pub Date : 2020-01-21 , DOI: 10.1016/j.pep.2020.105571
Dong Yao 1 , Jia Fan 1 , Ruizhi Han 1 , Jing Xiao 2 , Qian Li 3 , Guochao Xu 1 , Jinjun Dong 1 , Ye Ni 1
Affiliation  

Sucrose phosphorylase (SPase, EC 2.4.1.7) has a wide range of application in food, cosmetics, and pharmaceutical industries because of its broad substrate specificity. However, low SPase yields produced by wild-type strains cannot meet industrial requirements due to their complex metabolic regulation mechanisms. In this study, spase gene from Thermoanaerobacterium thermosaccharolyticum was cloned and expressed in Escherichia coli BL21 (DE3), leading to 7.05 U/mL (3.71 U/mg) of T. thermosaccharolyticum SPase (TtSPase) under optimum conditions. Co-expression of molecular chaperone teams pGro7 (GroES-GroEL), pG-KJE8 (DnaK-DnaJ-GrpE and GroES-GroEL), and pG-TF2 (GroES-GroEL-Tig) significantly enhanced the TtSPase activities to 18.5 U/mg (59.2 U/mL), 9.52 U/mg (28.6 U/mL), and 25.7 U/mg (64.5 U/mL), respectively. Results suggested that GroES-GroEL chaperone combination could regulate protein folding processes and protect misfolded proteins from aggregation. The enzymatic characterization results showed that TtSPase had an optimal temperature of 60 °C and optimal pH of 6.5. In particular, it had high thermostability of T5030 = 67 °C and half-life (t1/2 at 70 °C) of 19 min. Furthermore, purified TtSPase was used for hydroquinone transglycosylation and 21% of molar production yield of α-arbutin was obtained. This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli.

中文翻译:

通过分子伴侣增强大肠杆菌中蔗糖磷酸化酶的可溶性表达。

蔗糖磷酸化酶(SPase,EC 2.4.1.7)由于其广泛的底物特异性而在食品,化妆品和制药行业中具有广泛的应用。然而,由于其复杂的代谢调节机制,野生型菌株产生的低SPase产量不能满足工业要求。在这项研究中,来自嗜热解热厌氧杆菌的spase基因被克隆并在大肠杆菌BL21(DE3)中表达,从而在最佳条件下产生了7.05 U / mL(3.71 U / mg)的嗜热解球菌SPase(TtSPase)。分子伴侣团队pGro7(GroES-GroEL),pG-KJE8(DnaK-DnaJ-GrpE和GroES-GroEL)和pG-TF2(GroES-GroEL-Tig)的共表达显着提高了TtSPase活性至18.5 U / mg (59.2 U / mL),9.52 U / mg(28.6 U / mL)和25.7 U / mg(64.5 U / mL)。结果表明,GroES-GroEL分子伴侣组合可以调节蛋白质折叠过程,并防止错误折叠的蛋白质聚集。酶促表征结果表明,TtSPase的最佳温度为60°C,最佳pH为6.5。特别是,它具有T5030 = 67°C的高热稳定性和19分钟的半衰期(在70°C时为t1 / 2)。此外,将纯化的TtSPase用于氢醌转糖基化,并且获得21%的α-熊果苷的摩尔产率。这项研究为潜在的工业应用提供了具有高热稳定性的TtSPase,并开发了一种提高大肠杆菌中可溶性TtSPase产量的有效策略。酶促表征结果表明,TtSPase的最佳温度为60°C,最佳pH为6.5。特别是,它具有T5030 = 67°C的高热稳定性和19分钟的半衰期(在70°C时为t1 / 2)。此外,将纯化的TtSPase用于氢醌转糖基化,并且获得21%的α-熊果苷的摩尔产率。这项研究为潜在的工业应用提供了具有高热稳定性的TtSPase,并开发了一种提高大肠杆菌中可溶性TtSPase产量的有效策略。酶促表征结果表明,TtSPase的最佳温度为60°C,最佳pH为6.5。特别是,它具有T5030 = 67°C的高热稳定性和19分钟的半衰期(在70°C时为t1 / 2)。此外,将纯化的TtSPase用于氢醌转糖基化,并且获得21%的α-熊果苷的摩尔产率。这项研究为潜在的工业应用提供了具有高热稳定性的TtSPase,并开发了一种提高大肠杆菌中可溶性TtSPase产量的有效策略。
更新日期:2020-01-22
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