Abstract
A heat-stable sarcosine oxidase produced by Bacillus sp. BSD-8 (SOX) had been studied and its complete gene sequence, which contained 1,164 bp nucleotides and encoded a protein of 387 amino acids, was obtained by DNA Walking method. The sox gene was cloned and functionally overexpressed in E. coli and the recombinant SOX (rSOX) was purified to homogeneity, its properties was studied and compared with the wild type of SOX. The rSOX as well as SOX was stable at 60°C and at pH 7.0∼10.0, respectively. The optimal temperature for this enzyme was 60°C and at pH 8.5, it showed its highest activity. The Km and Kcat of the enzyme was 3.1 mM and 20.3/s, respectively. The difference between the properties of the SOX and rSOX was that the SOX contained noncovalent FAD, whereas the rSOX contained covalent FAD. The study also showed that an increased number of alanine residues in the rSOX might have some contribution in the enzymatic thermostability.
Similar content being viewed by others
References
Argos P, Rossmann MG, Grau UM, Zuber H, Frank G, Tratschin JD (2006) Thermal stability and protein structure. Biochemistry 18:5698–5703
Bacon BL, Pardue HL (1991) Predicitive error-compensating kinetic method for enzymetic quantification of creatinine in serum. Clin Chem 37:1338–1344
Borgi MA, Srih-Belguith K, Ben M, Mezghani M, Tranier S, Haser R, Bejar S (2004) Glucose isomerase of the Streptomyces sp. SK strain: purification, sequence analysis and implication of alanine 103 residue in the enzyme thermostability and acidotolerance. Biochimie 86:561–568
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein. Anal Biochem 72:248–254
Branden C, Tooze J (1991) Introduction to protein structure. Garland, New York
Chikayama M, Ohsumi M, Yokota S (2000) Enzyme cytochemical localization of sarcosine oxidase activity in the liver and kidney of several mammals. Histochem Cell Biol 113:489–495
Chlumsky LJ, Zhang L, Ramsey AJ, Jorns MS (1993) Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine. Biochemistry 32:11132–11142
Dao-Pin S, Baase WA, Matthews BW (1990) A mutant T4 lysozyme (Val 131 Ala) designed to increase thermostability by the reduction of strain within an alpha-helix. Proteins 7:198–204
Goyert A, Johnson TL, Olsen LJ, Collakova E, Hill YS, Rhodes D, Hanson AD (2004) Characterization and metabolic function of a peroxisomal sarcosine and pipecolate oxidase from Arabidopsis. J Biol Chem 279:16947–16953
Ichikawa T, Suzuki M, Koyama T (1993) Japan Patent 1993115281-A1
Imanaka T, Shibazaki M, Takagi M (1986) A new way of enhancing the thermostability of proteases. Nature 324:695–697
Kayama Y, Yamamoto H, Suzuki M, Nakano E (1991) Cloning and espression of the sarcosine oxidase gene from Bacillus sp.NS-129 in Escherichia coli. Agric Biol Chem 55:1259–1263
Kim JM, Shimizu S, Yamada H (1987) Crystallization and characterization of sarcosine oxidase from Alcaligenes denitrificans subp.denitrificans. Agric Biol Chem 51:1167–1168
Kinoshita T, Hiraga Y (1980) A fluorophotometric determination of serum creati-nine and creatine using a creatine amidohydrelase–creatine amidinohydrolase–sarcosine oxidase–peroxidase system and diacetyldichloroflurorescin. Chem Pharm Bull 28:3501–3506
Lim JH, Hwang KY, Choi J, Lee DY, Ahn BY, Cho Y, Kim KS, Han YS (2001) Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability. Biochem Biophys Res Commun 288: 263–268
Lindback B, Bergman A (1989) A new commercial method for the enzymetic determination of creatine in serum and urine evaluated: comparision with the kinetic Jaffe method and isotope dilution-mass spectrometry. Clin Chem 25:835–837
Matsuda Y, Hoshika H, Inouye Y, Ikut S, Matsuura K, Nakamura S (1987) Purification and characterization of sarcosine oxidase of Bacillus origin. Chem Pharm Bull 35:711–717
Menendez-Arias L, Argos P (2006) Engineering protein thermal stability: sequence statistics point to residue substitutions in a-helices. J Mol Biol 206:397–406
Mori N, Sano M, Tani Y, Yamda H (1980) Purification and properties of sarcosine oxidase from Cylindrocarpon didymium M-1. Agric Biol Chem 44:1391–1397
Nishiya Y, Imanaka T (1993) Cloning and sequencing of the sarcosine oxidase gene from Arthrobacter sp.TE1826. J Ferment Bioeng 75:239–244
Ogushi S, Nagao K, Emi S, Ando M, Tsuru D (1988) Sarcosine oxidase from Arthrobacter ureafaciens: purification and some properties. Chem Pharm Bull 36:1445–1450
Parajuli N, Basnet DB, Chung YS, Lee HC, Liou K, Liou K (2005) Biochemical characterization of a novel thermostable glucose-1-phosphate thymidylyltransferase from Thermuscaldophilus: probing the molecular basis for its unusual thermostability. Enzyme MicrobTechnol 37:402–409
Peter B, Josef A, Klaus DK, Ralf M (1997) Cloning, nucleotide sequence and expression of a mannitol dehydrogenase gene from Pseudomonas fluorescens DSM 50106 in Escherichia coli. Biochim Biophys Acta 1351:157–167
Reuber BE, Karl C, Reimanm SA, Mihalik SJ, Dodt G (1997) Cloning and functional expression of a mammalian gene for a peroxisomal sarcosine oxidase. J Biol Chem 272:6766–6776
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Sanger F, Nicklen S, Coulson AR (1997) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci 74:5463–5467
Slebert PD, Chenchik A, Kellogg DE (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088
Suzuki M (1981) Purification and some properties of sarcosine oxidase from Corynebacterium sp. U-96. J Biochem 89:599–607
Suzuki M (1988) US Patent 4,740,465
Suzuki H (1994) Sarcosine oxidase: structure, function, and the application to creatinine determination. Amino Acids 7:27–43
Suzuki K, Ogishima M, Sugiyama M, Inouye Y, Nakamura S, Imamura S (1992) Molecular cloning and expression of a Streptomyces sarcosine oxidase gene in Streptomyces lividans. Biosci Biotechnol Biochem 56:432–436
Suzuki K, Sagai H, Sugiyama M (1994) Cloning, sequencing and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene. J Ferment Bioeng 77:231–234
Trichey P, Wagner MA, Jorns MS, Mathews FS (1999) Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme. Structure Fold Des 7:331–345
Vetriani C, Maeder DL, Tolliday N, Yip KS, Stillman TJ, Britton KL, Rice DW, Klump HH, Robb FT (1998) Protein thermostability above 100°C: a key role for ionic interactions. Proc Natl Acad Sci USA 95:12300–12305
Willie A, Jorns MS (1995) Discovery of a third coenzyme in sarcosine oxidase. Biochemistry 34:16703–16707
Willie A, Edmondson DE, Jorns MS (1996) Sarcosine oxidase contains a novel covalently bound FMN. Biochemistry 35:5292–5299
Yutani K, Ogasahara K, Tsujita T, Sugino Y (1987) Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase subunit. Proc Natl Acad Sci USA 81:4441–4444
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Guo, K., Ma, X., Sun, G. et al. Expression and characterization of a thermostable sarcosine oxidase (SOX) from Bacillus sp. in Escherichia coli . Appl Microbiol Biotechnol 73, 559–566 (2006). https://doi.org/10.1007/s00253-006-0502-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-006-0502-7