由于目前大部分肌氨酸氧化酶(sacrosine oxidase, SOX)热稳定性差、失活机理研究不深入,使其在医疗和食品工业等领域的实际应用中受限。该研究通过筛选与挖掘一种热稳定性优异的肌氨酸氧化酶,以达到拓宽其应用前景与应用潜力的目的。根据预测,将来源于古生菌Archaea HR32中肌氨酸氧化酶在Bacillus subtilis WB600中异源表达,并对其酶学性质进行考察。重组蛋白rSOX大小约为42 kDa,二级结构由35.0% α-螺旋、11.1% β-折叠、23.3% β-转角及31.6%无规则卷曲组成,Tm和ΔH为92.13 ℃和1 070 kJ/mol。以肌氨酸为底物时,最适pH为8.0,最适温度为70 ℃,80 ℃下半衰期为12 h,Km、kcat和kcat/Km分别为1.17 mmol/L、74.59 min-1、63.78 L/(mmol· min)。常见有机溶剂对rSOX活性影响不大,金属离子则对rSOX有不同程度的促进或抑制作用。另外,rSOX还可催化降解其他多种N-甲基类化合物,且对N-甲基-L-型底物有明显的手性选择性。综上,该研究获得了一种来源于古生菌的新型耐热肌氨酸氧化酶,相关酶学性质实验为研究该酶对N-甲基类化合物的降解奠定理论基础,进而提升该类酶的应用潜力。
Due to the relatively poor thermostability and the lack of comprehensive understanding of the deactivation mechanism for sarcosine oxidase (SOX), the obstacle to the large-scale application of this type of enzyme is limited. In this study, the application potential of SOX is aimed to be enhanced by screening and isolating a novel SOX characterized with exceptional thermal stability. Via prediction, a predicted SOX from the genome of Archaea HR32 was recombinantly expressed in Bacillus subtilis WB600 and further characterized of the enzymatic properties. The purified rSOX was identified with a size of approximate 42 kDa and the secondary structure of it was observed to be composed of 35.0% α-helix, 11.1% β-sheet, 23.3% β-turn, and 31.6% random coil. The melting temperature (Tm) and the denaturation enthalpy (ΔH) were verified as 92.13 ℃ and 1 070 kJ/mol, respectively. When having sarcosine as the substrate, the optimal pH and temperature were characterized as 8.0 and 70 ℃. With the half-time of 12 h at 80 ℃, the Km, kcat and kcat/Km of rSOX were measured as 1.17 mmol/L, 74.59 min-1 and 63.78 L/(mmol· min), respectively. rSOX was also observed to exhibit resistance to normal organic solvents, whilst metal ions were found to have either promotion or inhibition effect on the enzymatic activity. In addition, the current research verified the catalytic degradation capability of rSOX to different types of N-methyl compounds, in particular the chiral selectivity against the L-type substrates. Overall, with screening and obtaining of a newly SOX with extensive thermal stability from Archaea, the enzymatic properties was investigated to provide theoretical foundation for its further utilization in the field of N-methyl substrates degradation and hence to further improve the application potential for this type of enzyme.
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