Thermomicrobium roseum sarcosine oxidase (TrSOX) is an N-demethylase with specific substrate chiral selectivity, good resistance against high temperature and organic solvents. TrSOX catalyze the methyl removal reaction of sarcosine to produce glycine, with the cofactor flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). In the food industry, sarcosine-based oxidation biosensors have been widely used for the determination of organic acids and glycerol in wine fermentation. Arg54 is one of the conserved residues in the catalytic center, in order to investigate the effects of this site on the enzymatic properties and to screen out mutants with good catalytic efficiency, site-saturation mutagenesis at ArgLys54 was carried out in a pre-constructed xylose-induced plasmid pMA5-Pxyl-trsox, and the resulted plasmids were transformed to Bacillus subtilis WB600 for expression. All 15 of the 19 mutants could be expressed and prepared except for R54S, R54C, R54H, R54T and R54W,because plasmids carrying the four mutant genes mentioned above are unable to express soluble and active enzyme molecules in the host cell. Determination of the protein concentration of the crude enzyme solution revealed an increase in total protein for R54D, R54K, R54Q, R54V and an approximately 2.5-fold increase for R54D. The analysis of the properties of mutant TrSOX and natural TrSOX showed that mutant TrSOX maintained the excellent thermal stability of natural TrSOX, however, the secondary structures of most mutants were affected except R54Q, R54N, R54K, R54D and R54V. The reason for the change in secondary structure of most mutants may be that the ArgLys54 site is more important for the maintenance of the overall structure of TrSOX and its mutation affects the overall folding of the protein structure. Although the secondary structure of most of the mutants was affected, the thermal stability was not greatly affected, because the regions where the secondary structure was altered did not affect the unchaining temperature of the mutants and did not make a difference to the energy used to disrupt the overall structure of the protein, resulting in the mutants continuing the excellent thermal stability of natural TrSOX. In addition, the chiral specificity of mutants for substrates were same as those of the wild TrSOX; The mutants could specifically recognize N-methyl-L-amino acids and could deplete the methyl group, but no catalytic effect on the D-conformation. A comparative study of the catalytic activity of these mutants revealed that the catalytic efficiency of R54D and R54K for N -methyl- L-amino acids was significantly increased, while the other mutants showed reduced efficiency or even complete inactivation. For the basic residue mutant R54K, the catalytic efficiency was increased by 3.17-fold, 5.99-fold, 9.12-fold, 7.61-fold, 1.00-fold and 2.07-fold for N-methyl-glycine, N-methyl-L-alanine,N-methyl-L-phenylalanine, N-methyl-L-leucine,N-methyl-L-tryptophan and N-methyl-L-aspartic acid respectively. The mutant R54D containing acidic residues also showed a significant increase in the catalytic efficiency of N-methyl-L-amino acids, with 1.79-fold, 5.57-fold, 17.92-fold, 9.00-fold and 1.43-fold increases in the catalytic efficiency of N-methyl-glycine, N-methyl-L-alanine, N-methyl-L-phenylalanine, N-methyl-leucine and N-methyl-L-tryptophan, respectively .The catalytic effect of mutant TrSOX on N-methyl-L-amino acid substrates lies in the fact that the basic or acidic side chain of the R54 residue can interact directly with the active side chain of the substrate, directing the entry of the substrate and the release of the product, thereby enhancing catalytic properties. In addition, for non-amino acid substrates, natural TrSOX was inactive against all substrates except L-homoproline, and mutants such as R54I,R54Q,R54N,R54G,R54K,R54P,R54D showed catalytic activity not present in wild-type TrSOX when substrated with alkaloids such as pyrrolidine ((3S)-(+)-3-(methylamino)pyrrolidine), piperidine (piperidine and 2-methylpiperidine), even larger structures (caprolactam) or more complex ones (1,2,3,4-tetrahydroisoquinoline and 1-methyl-1,2,3,4-tetrahydroisoquinoline) as substrates, catalytic activity was detected that was not present in wild-type TrSOX. This suggests that the basic guanidino side chain of R54 may reject these substrates, which in the above mutant can enter the active pocket and be converted to the relevant product. The catalytic mechanism of mutant TrSOX for non-amino acid substrates is similar to the original catalytic mechanism, but further studies (LC-MS/NMR) are needed to clarify the products. However, the catalytic efficiency of these non-amino acid bound substrates is much lower than that of N-methylamino acids and, in addition, the catalytic mechanism and products of the non-amino acids are not yet known. In our further work, there is a need to further research the catalytic ability for such substrates and to purify and characterize the products.
[1] 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.
[2] DIJKMAN W P, DE GONZALO G, MATTEVI A, et al.Flavoprotein oxidases:Classification and applications[J].Applied Microbiology and Biotechnology, 2013, 97(12):5 177-5 188.
[3] BACON B L, PARDUE H L.Predictive, error-compensating kinetic method for enzymatic quantification of creatinine in serum[J].Clinical Chemistry, 1991, 37(8):1 338-1 344.
[4] MONOŠÍK R, UKROPCOVÁ D, STREDANSKÝ 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.
[5] 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.
[6] TONG Y J, FENG S S, XIN Y, et al.Enhancement of soluble expression of codon-optimized Thermomicrobium roseum sarcosine oxidase in Escherichia coli via chaperone co-expression[J].Journal of Biotechnology, 2016, 218:75-84.
[7] 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.
[8] TONG Y J, XIN Y, YANG H L, et al.Efficient improvement on stability of sarcosine oxidase via poly-lysine modification on enzyme surface[J].International Journal of Biological Macromolecules, 2014, 67:140-146.
[9] MENG D D, LIANG A L, WEI X L, et al.Enzymatic characterization of a thermostable phosphatase from Thermomicrobium roseum and its application for biosynthesis of fructose from maltodextrin[J].Applied Microbiology and Biotechnology, 2019, 103(15):6 129-6 139.
[10] 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.
[11] NIU C T, LIU C F, LI Y X, et al.Production of a thermostable 1, 3-1, 4-β-glucanase mutant in Bacillus subtilis WB600 at a high fermentation capacity and its potential application in the brewing industry[J].International Journal of Biological Macromolecules, 2018, 107:28-34.
[12] WU X C, LEE W, TRAN L, et al.Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases[J].Journal of Bacteriology, 1991, 173(16):4 952-4 958.
[13] 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.
[14] GAO Q Y, SHAO J, TANG M W, et al.Promote the expression and corrected folding of an extremely stable N-demethylase by promoter reconstruction, native environment simulation and surface design[J].International Journal of Biological Macromolecules, 2021, 178:434-443.
[15] 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,CN111111085A[P].2020-05-18.
XIN Y,ZHANG L.Application of recombinant Bacillus subtilis cells in enzymatic biodegradation of N-methyl organic nitrogen pesticides:China, CN111111085A[P].2021-05-18.
[16] SELFRIDGE B R, WANG X H, ZHANG Y N, et al.Structure-activity relationships of (+)-naltrexone-inspired toll-like receptor 4 (TLR4) antagonists[J].Journal of Medicinal Chemistry, 2015, 58(12):5 038-5 052.
[17] KOK G, ASHTON T, SCAMMELLS P.An improved process for the N-demethylation of opiate alkaloids using an iron(II) catalyst in acetate buffer[J].Advanced Synthesis & Catalysis, 2009, 351(1-2):283-286.
[18] KIM J H, KIM B H, BROOKS S, et al.Structural and mechanistic insights into caffeine degradation by the bacterial N-demethylase complex[J].Journal of Molecular Biology, 2019, 431(19):3 647-3 661.
[19] AUGUSTIN M M, AUGUSTIN J M, BROCK J R, et al.Enzyme morphinan N-demethylase for more sustainable opiate processing[J].Nature Sustainability, 2019, 2(6):465-474.
[20] 辛瑜, 张梁, 张瑶, 等.一种酶法拆分制备N-甲基-D-天冬氨酸的方法:中国,CN109628519A[P].2019-04-16.
XIN Y,ZHANG L,ZHANG Y,et al.A kind of method for preparing N-methyl-D-aspartic acid by enzymatic splitting:China, CN109628519A[P] 2019-04-16.
[21] SONG B W, CHEN K Q, TANG Y J, et al.ConsRM:collection and large-scale prediction of the evolutionarily conserved RNA methylation sites, with implications for the functional epitranscriptome[J].Briefings in Bioinformatics, 2021, 22(6):bbab088.
[22] UCHIDA T, DOJUN N, OTA K, et al.Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction[J].Archives of Biochemistry and Biophysics, 2019, 677:108165.
[23] LI X H, SUN J J, WANG W, et al.Site-saturation mutagenesis of proline 176 in cyclodextrin glucosyltransferase from Bacillus sp.Y112 effects product specificity and enzymatic properties[J].Process Biochemistry, 2020, 94:180-189.
[24] KE T, LIU J, ZHAO S, et al.Using global transcription machinery engineering (GTME) and site-saturation mutagenesis technique to improve ethanol yield of Saccharomyces cerevisiae[J].Applied Biochemistry and Microbiology, 2020, 56(5):563-568.
[25] 季文明, 陈毅力, 张和春, 等.比色法测定胆固醇氧化酶酶活[J].无锡轻工大学学报, 2000, 19(3):251-254.
JI W M, CHEN Y L, ZHANG H C, et al.Colorimetric determination of the activity of cholesterol oxidase[J].Journal of Wuxi University of Light Industry, 2000, 19(3):251-254.
