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.
[1] CALISKAN S, YILDIRIM E, ANAKOK A, et al.Design of a new biosensor platform for creatinine determination[J].Journal of Solid State Electrochemistry, 2022:1-9.
[2] ZERAVIK J, FOHLEROVA Z, MILOVANOVIC M, et al.Various instrumental approaches for determination of organic acids in wines[J].Food Chemistry, 2016, 194:432-440.
[3] MONOŠÍK R, UKROPCOVÁ D, STREĎANSKÝ M, et al.Multienzymatic amperometric biosensor based on gold and nanocomposite planar electrodes for glycerol determination in wine[J].Analytical Biochemistry, 2012, 421(1):256-261.
[4] 邓永飞, 刘涛, 吴海铨, 等.食品工业废水处理技术研究进展[J].工业水处理, 2021, 41(10):1-7;13.
DENG Y F, LIU T, WU H Q, et al.Research progress of wastewater treatment technology in food industry[J].Industrial Water Treatment, 2021, 41(10):1-7;13.
[5] MATSUDA Y, HOSHIKA H, INOUYE Y, et al.Purification and characterization of sarcosine oxidase of Bacillus origin[J].Chemical and Pharmaceutical Bulletin, 1987, 35(2):711-717.
[6] SUZUKI M.Purification and some properties of sarcosine oxidase from Corynebacterium sp.U-96[J].The Journal of Biochemistry, 1981, 89(2):599-607.
[7] 辛瑜,张梁.一种重组枯草芽孢杆菌细胞在酶法生物降解N-甲基有机氮农药中的应用:中国,CN202010020741.1[P].2020-05-08.
XIN Y, ZHANG L.Application of recombinant subcorporea cells in enzymatic biodegradable N-methyl organic nitrogen pesticides:China, CN202010020741.1[P].2020-05-08.
[8] TURNER P, MAMO G, KARLSSON E N.Potential and utilization of thermophiles and thermostable enzymes in biorefining[J].Microbial Cell Factories, 2007, 6(1):1-23.
[9] XIN Y, HAO M Y, FAN G M, et al.Soluble expression of Thermomicrobium roseum sarcosine oxidase and characterization of n-demethylation activity[J].Molecular Catalysis, 2019, 464:48-56.
[10] SAITO M, ITOH A, SUZUKI H.Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96:The anionic form of the substrate in the enzyme-substrate complex is a reactive species[J].The Journal of Biochemistry, 2012, 151(6):633-642.
[11] HASSAN-ABDALLAH A, ZHAO G H, ESCHENBRENNER M, et al.Cloning, expression and crystallization of heterotetrameric sarcosine oxidase from Pseudomonas maltophilia[J].Protein Expression and Purification, 2005, 43(1):33-43.
[12] INOUYE Y, NISHIMURA M, MATSUDA Y, et al. Purification and characterization of sarcosine oxidase of Streptomyces origin[J].Chemical & Pharmaceutical Bulletin, 1987, 35(10):4 194-4 202.
[13] WANG Q, XIN Y, YANG H, et al.Deflavination of Bacillus sp. sarcosine oxidase and reconstitution of flavoproteins[J].Journal of Food Science and Biotechnology, 2018, 37(9):909-914.
[14] LIU H, SUN G, MA X, et al.Purification and characterization of a sarcosine oxidase from Bacillus sp. BSD-8[J].Chinese Journal of Biotechnology, 2010, 26(3):335-340.
[15] OGUSHI S, NAGAO K, EMI S, et al.Sarcosine oxidase from Arthrobacter ureafaciens:Purification and some properties[J].Chemical and Pharmaceutical Bulletin, 1988, 36(4):1 445-1 450.
[16] 郑梦玲. 嗜热玫瑰红球菌肌氨酸氧化酶表达、结构及功能研究[D].无锡:江南大学, 2018.
ZHENG M L.Study on expression, structure and function of Thermomicrobium roseum SOX[D].Wuxi:Jiangnan University, 2018.
[17] GUO K P, MA X H, SUN G Q, et al.Expression and characterization of a thermostable sarcosine oxidase (SOX) from Bacillus sp.in Escherichia coli[J].Applied Microbiology and Biotechnology, 2006, 73(3):559-566.
[18] SCHRIEVER K, SAENZ-MENDEZ P, RUDRARAJU R S, et al.Engineering of ancestors as a tool to elucidate structure, mechanism, and specificity of extant terpene cyclase[J].Journal of the American Chemical Society, 2021, 143(10):3 794-3 807.
[19] KIM J M, SHIMIZU S, YAMADA H.Crystallization and characterization of sarcosine oxidase from Alcaligenes denitrificans subsp. denitrificans[J].Agricultural and Biological Chemistry, 1987, 51(4):1 167-1 168.
[20] PUNDIR C, CHAUHAN N, KUMARI G, et al.Immobilization of Arthrobacter sarcosine oxidase onto alkylamine and arylamine glass and its application in serum sarcosine determination[J].Indian Journal of Biotechnology, 2011, 10(2):219-223.
[21] XIN Y, ZHENG M, WANG Q, et al.Structural and catalytic alteration of sarcosine oxidase through reconstruction with coenzyme-like ligands[J].Journal of Molecular Catalysis B:Enzymatic, 2016, 133:S250-S258.
[22] 林荣, 宋祖坤, 张玲, 等.FAD为辅基的葡萄糖脱氢酶发酵、纯化及酶学性质[J].食品与生物技术学报, 2020, 39(2):104-111.
LIN R, SONG Z K, ZHANG L, et al.Enzyme characterization, fermentation and purification for FAD-conjugated glucose dehydrogenase[J].Journal of Food Science and Biotechnology, 2020, 39(2):104-111.
[23] ZHENG M L, HAO M Y, FAN G M, et al.Preparation, reconstruction, and characterization of a predicted Thermomicrobium roseum sarcosine oxidase[J].Molecular Catalysis, 2018, 455:132-142.
[24] GAO J X, OU X Y, XU P, et al.Cloning, overexpression, and characterization of a novel organic solvent-tolerant lipase from Paenibacillus pasadenensis CS0611[J].Chinese Journal of Catalysis, 2018, 39(5):937-945.
[25] 仝艳军, 辛瑜, 杨海麟, 等.肌氨酸氧化酶的酶学性质及失活机理[J].食品与生物技术学报, 2015, 34(12):1 239-1 247.
TONG Y J, XIN Y, YANG H L, et al.On the enzymatic properties and deactivation mechanism of sarcosine oxidase[J].Journal of Food Science and Biotechnology, 2015, 34(12):1 239-1 247.
[26] 高秋月. 嗜热玫瑰红球菌肌氨酸氧化酶的表达优化及催化Loop域的改造研究[D].无锡:江南大学, 2021.
GAO Q Y.Optimization of expression and modification of the catalytic loop domain of Thermomicrobium roseum sarcosine oxidase[D].Wuxi:Jiangnan University, 2021.
