Abstract
The trehalose synthase (TreS) from Pseudomonas putida ATCC47054 was highly expressed in E.coli BL21(DE3) using pET15b and pET22b as expression vector. The expression of TreS protein in recombinant E.coli BL21(DE3)/pET15b-treS reached 34.3% of total protein, is better than E.coli BL21(DE3)/pET22b-treS reached 19.9%. The optimum pH and temperature of purified TreS by Ni2+ affinity chromatography is 8.0 and 25 ℃, and stable between pH 6.5 to 9.0, and exhibited the thermalstability from 15 to 40 ℃. When the conversion system temperature of TreS is more than 40 ℃, enzyme activity will accelerate loss. But a special phenomenon was discovered that high substrate maltose concentration is advantageous to the thermal stability of enzyme activity. When the substrate maltose concentration reached 300 g/L, the TreS still has a stable conversion efficiency at 50 ℃. In order to improve the intracellular expression of TreS, the fermentation and induction conditions of recombinant E.coli BL21(DE3)/pET15b-treS were optimized. Using final concentration 4 g/L lactose as inducing agent to induce 7 h at 27 ℃ in 5-L fermentor, the enzyme activity reached 39,866 ± 1420 U per gram dry cell weight. For avoid the cost of enzyme purification, the cell lysis solution was directly used to convert high maltose syrups for preparation of trehalose, and over 64% of maltose in conversion system can be converted into trehalose with substrate maltose concentration 300 g/L at 50 ℃ and pH 8.0.
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Elbein AD, Pan YT, Pastuazak I, Carroll D (2003) New insights on trehalose: a multi-functional molecule. Glycobiology 13(4):17–27
Whatmore AM, Reed RH (1990) Determination of turgor pressure in Bacillus subtilis: a possible role for K+ in turgor regulation. J Gen Microbiol 136(12):2521–2526
Carpinelli J, Kraemer R, Agosin E (2006) Metabolic engineering of Corynebacterium glutamicum for trehalose over production: role of the TreYZ trehalose biosynthetic pathway. Appl Environ Microbiol 72(3):1949–1955
Murphy HN, Stewart GR, Mischenko VV, Apt AS, Harris R, McAlister MS, Driscoll PC, Young DB, Robertson BD (2005) The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis. J Biol Chem 280(15):14524–14529
Higashiyama T (2002) Novel functions and applications of trehalose. Pure Appl Chem 74(7):1263–1269
Kempf B, Bremer E (1998) Uptake and synthesis of compatible solutes as microbial sTreSs responses to high-osmolality environments. Arch Microbiol 170(5):319–330
Rueda B, Miguelez EM, Hardisson C, Manzanal MB (2001) Changes in glycogen and trehalose content of Streptomyces brasiliensis during growth in liquid cultures under sporulating and non-sporulating conditions. FEMS Microbiol Lett 194(2):181–185
Crowe LM (2002) Lessons from nature: the role of sugar in anhydrobiosis. Com Biochem Physio A-molecular Integr Physiol 131(3):505–513
Duong T, Barrangou R, Russell WM, Klaenhammer TR (2006) Characterisation of the tre locus and analysis of trehalose cryoprotection in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 72(2):1218–1225
Rao V, Gao F, Chen B, Jacobs WR Jr, Glickman MS (2006) Trans-cyclopropanation of mycolic acids on trehalose dimycolate suppresses Mycobacterium tuberculosis induced inflammation and virulence. J Clin Invest 116(6):1660–1667
Schwendeman SP, Constantino HR, Gupta RK, Siber GR, Klibanov AM, Langer R (1995) Stabilization of tetanus and diphtheria toxoids against moisture induced aggregation. Proc Natl Acad Sci USA 92(24):11234–11238
Jain NK, Roy I (2008) Role of trehalose in moisture induced aggregation of bovine serum albumin. Eur J Pharm Biopharm 69(3):824–834
Crowe JH, Leslie SM, Crowe LM (1994) Is vitrification sufficient to preserve liposomes during freeze drying. Cryobiology 31(4):355–366
Guo N, Puhlev I, Brown DR, Mansbridge J, Levine F (2000) Trehalose expression confers desiccation tolerance on human cells. Nat Biotechnol 18(2):168–171
Eroglu A, Russo MJ, Bieganski R, Fowler A, Cheley S, Bayley H, Toner M (2000) Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat Biotechnol 18(2):163–167
Eroglu A, Toner M, Toth TL (2002) Beneficial effect of microinjected trehalose on the cryosurvival of human oocytes. Fertil Steril 77(1):152–158
Lee JH, Lee KH, Kim CG, Lee SY, Kim GJ, Park YH, Chung SO (2005) Cloning and expression of aTreS from Pseudomonas stutzeri CJ38 in Escherichia coli for the production oftrehalose. Appl Microbiol Biotechnol 68:213–219
Yue M, Wu XL, Gong WN, Ding HB (2009) Molecular cloning and expression of a novel TreS gene from Enterobacter hormaechei. Microb Cell Fact 8:34
Wu X, Ding H, Yue M, Qiao Y (2009) Gene cloning, expression, and characterization of a novel TreS from Arthrobacter aurescens. Appl Microbiol Biotechnol 83(3):477–482
Kim TK, Jang JH, Cho HY, Lee HS, Kim YW (2010) Gene cloning and characterization of a TreS from Corynebacterium glutamicum ATCC13032. Food Sci. Biotechnol 19(2):565–569
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Liu, HL., Wang, RM., Wang, TF. (2018). High Efficiency Expression of Trehalose Synthase in Escherichia coli and Its Use in the Production of Trehalose. In: Liu, H., Song, C., Ram, A. (eds) Advances in Applied Biotechnology. ICAB 2016. Lecture Notes in Electrical Engineering, vol 444. Springer, Singapore. https://doi.org/10.1007/978-981-10-4801-2_5
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DOI: https://doi.org/10.1007/978-981-10-4801-2_5
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