Expression and characterization of recombinant trehalose synthase in <i>Bacillus subtilis<\i>
Keywords:Bacillus subtilis, cloning, expression of recombinant protein, enzyme characteristics, trehalose synthase.
Trehalose synthase (TreS, EC 22.214.171.124) is a potential catalyst for synthesis of trehalose, an important natural disaccharide. In this study, the treS gene of Pseudomonas putida (VTCC 12263) was cloned into pHT01 plasmid at BamHI-XbaI position, expressed in Bacillus subtilis (B. subtilis) 1012, and characterized. The recombinant TreS had molecular weight of 68 kDa when fused with 8xHis tag at the C-terminus. catalyzed conversion of maltose to trehalose in optimal conditions had specific activity of 1.664 U/g. Expression of TreS was highest when B. subtilis 1012 harboring pHT01-treS was cultured in TB medium at 30 oC, induced with 1.0 mM IPTG when OD600 reached 0.8 and harvested after 10 hours of induction. The recombinant TreS purified by Ni-sepharose chromatography had specific activity of 41.700 U/g and formed a single band on Western blot with monoclonal antibody against His-tag. The recombinant TreS had optimal activity at 37 oC in 100 mM pH 7.4 PBS and 300 mM maltose. It was inhibited by NaCl, KCl and MgCl2 (retaining 45% or 75% specific activity in buffer containing 5 mM KCl or 5 mM MgCl2, respectively) and stimulated by imidazol (with specific activity increasing by 30–200%).
Chen Y. S., Lee G. C., Shaw J. F., 2006. Gene Cloning, Expression, and Biochemical Characterization of a Recombinant Trehalose Synthase from Picrophilus torridusin in Escherichia coli. J. Agric. Food Chem., 54: 7098–7104.
Kim T. K., Jang J. H., Cho H. Y., Lee H. S., Kim Y. W., 2010. Gene cloning and characterization of a trehalose synthase from Corynebacterium glutamicum ATCC13032. Food Sci. Biotechnol., 19: 565–569.
Lee J. H., Lee K. H., Kim C. G., Lee S. Y., Kim G. J., Park Y. H., Chung S. O., 2005. Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose. Appl. Microbiol. Biotechnol., 68: 213–219.
Li Y., Gu Z., Zhang L., Ding Z., Shi G., 2016. Inducible expression of trehalose synthase in Bacillus licheniformis. Protein Expr. Purif., http://doi.org/10.1016/j.pep.2016. 10.005
Lim H. K., Lee S. U., Chung S. I., Ung K. H., Seo J. H., 2004. Induction of the T7 Promoter Using Lactose for Production of Recombinant Plasminogen Kringle 1−3 in Escherichia coli. J. Microbiol. Biotechnol. 14: 225–230.
Liu H., Yang S., Wang X., Wang T., 2019. Production of trehalose with trehalose synthase expressed and displayed on the surface of Bacillus subtilis spores. Microb. Cell Fact., 18: 1–13.
Ma Y., Xue L., Sun D.W. 2006., Characteristics of trehalose synthase from permeablized Pseudomonas putida cells and its application in convertin maltose into trehalose. J. Food. Eng., 77: 342–347. https://doi.org/10.1016/ j.jfoodeng.2005.06.042
Mahmood T., Yang P. C. 2012., Western blot: technique, theory, and trouble shooting. N. Am. J. Med. Sci., 4: 429–434.
Nogales J., Palsson B., Thiele I., 2008. A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory. BMC Systems Biology, 2: 1–20. https://doi.org/10.1186/1752-0509-2-79
Nguyen H. D., Phan T. T. P., Schumann W., 2007. Expression vectors for the rapid purification of recombinant proteins in Bacillus subtilis. Current Microbiology, 55: 89–93.
Nguyen T. H. T., Nguyen T. T., Le T. H., Nguyen T. A. T., Nguyen M. D., Le D. M., Do T. T., 2018. Screening bacterial strains for production of maltooligosyl trehalose trehalohydrolase and maltooligosyl trehalose synthase. Journal of Vietnamese Environment, 9(2): 55–60.
Phan T., Huynh P., Truong T., Nguyen H., 2017. A generic protocol for intracellular expression of recombinant protein in Bacillus subtilis. Methods Mol. Biol., 1586: 325–334.
Sarvas M., 1995. Gene expression in recombinant Bacillus. Bioprocess. Technol., 22: 53–120.
Wang T., Jia S., Dai K., Liu H., Wang R., 2014. Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose. Canadian Journal of Microbiology, 60(9): 599–604. https://doi.org/10.1139/ cjm-2014-0330
Xiuli W., Hongbiao D., Ming Y., Yu Q., 2009. Gene cloning, expression, and characterization of a novel trehalose synthase from Arthrobacter aurescens. Applied microbiology and biotechnology, 83(3): 477–482. https://doi.org/ 10.1007/s00253-009-1863-5
Yan J., Qiao Y., Hu J., Ding H., 2013. Cloning, expression and characterization of a trehalose synthase gene from Rhodococcus opacus. Protein J., 32: 223–229.
Yue M., Wu X. L., Gong W. N., Ding H. B., 2009. Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei. Microb. Cell Fact., 8: 34.