极端环境微生物是发掘性能优良新酶的重要资源,其中高稳定性的酶类在工业生产中具有巨大的潜在应用价值。该研究通过异源重组表达了嗜热玫瑰红球菌(Thermomicrobium roseum DSM 5159)来源的一段预测谷氨酰胺酶基因,并对其酶学性质进行了研究。利用镍柱亲和层析纯化表达产物,通过基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF-MS)鉴定纯化结果,分析了重组酶的催化功能,以正交试验优化了表达条件,考察了重组酶的酶学性质。结果表明,重组谷氨酰胺酶的二级结构由35.78% α-螺旋,14.07% β-折叠,16.06% β-转角以及34.19%无规则卷曲组成,热变性温度(Tm)和变性焓(ΔH)分别为94.38 ℃和1 672 kJ/mol,以L-谷氨酰胺为底物时,最适反应温度为70 ℃,最适反应pH值为9.0,70 ℃以下酶活力半衰期大于12 h,其耐热性能良好,动力学研究表明重组谷氨酰胺酶对L-谷氨酰胺的亲和力高于D-谷氨酰胺,Mn2+、Fe2+、Zn2+、Cu2+、Co2+对酶活力有明显抑制作用。对嗜热古生菌来源的谷氨酰胺酶的结构及酶学性质的研究为挖掘开发具有优良热稳定性的工业生产用酶类提供了一定的参考价值。
Microorganisms from extreme environment are valuable sources for discovering new enzymes with unique properties, particularly highly stable enzymes with significant potential for industrial applications.In this study, a segment of predicted glutaminase gene from Thermomicrobium roseum DSM 5159 source was expressed by heterologous recombination and its enzymatic properties were investigated.The expression product was purified using nickel column affinity chromatography, the purification results were identified by matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS), the catalytic function of the recombinant enzyme was analyzed, the expression conditions were optimized by orthogonal test, and the enzymatic properties of the recombinant enzyme were investigated.The results revealed that the secondary structure of the recombinant glutaminase consisted of 35.78% α-helix, 14.07% β-sheet, 16.06% β-turn, and 34.19% random coil.The thermal denaturation temperature (Tm) and enthalpy change (ΔH) were 94.38 ℃ and 1 672 kJ/mol, respectively.The optimal reaction temperature for using L-glutamine as the substrate was 70 ℃, and the optimal pH value was 9.0.The enzyme retained more than 50% of its activity after 12 hours at temperatures below 70 ℃, demonstrating excellent heat stamina.Kinetic studies indicated that the recombinant glutaminase had a higher affinity for L-glutamine than for D-glutamine.Additionally, Mn2+, Fe2+, Zn2+, Cu2+, and Co2+ significantly inhibited enzyme activity.The study of the structure and enzymatic properties of glutaminase from Thermophilic archaea provides a certain reference value for mining and developing enzymes with excellent thermal stability for industrial production.
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