Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose

Abstract

A novel strain was isolated, Pseudomonas stutzeri CJ38, that enabled direct transformation of maltose to trehalose. In comparison with others reported to date, CJ38 provided a novel trehalose synthase (TSase) without any byproduct, including glucose. Activity analysis, using either maltose or trehalose as a substrate, showed a reversible reaction. There was also no detectable activity of related enzymes with liquid starch and maltooligosaccharides as substrates. Using a malPQ-negative host and MacConkey medium, the TSase gene was cloned in Escherichia coli from CJ38. The resulting sequence contained an open reading frame consisted of 689 amino acids with a calculated molecular mass of 76 kDa. A search for related sequences in various gene and protein data banks revealed a novel family of enzymes that was predicted putatively as a glycosidase or TSase family, with no biochemical evidence. The recombinant enzyme exhibited a high activity toward the substrate maltose, about 50-fold higher than the parent strain and resulted in a high conversion yield (72%) at a relatively high substrate concentration (20%). These results provided the possibility that the strain was effectively used as a potential biocatalyst for the production of trehalose from maltose in a one-step reaction.

Characterization and Identification of The Key Residues Abound The Activity Site Tunnel of Trehalose Synthase from Pseudomonas Stutzeri Qlu3

“…2E, approximately 70% of the maltose substrate was converted to trehalose after a 1-hr reaction. The reaction time was obviously shorter than previously reported [10]. The glucose byproduct yield was low, at 3.2%.…”

“…Thus, TreS is considered as a convenient and practical biocatalyst for the industrial production of trehalose because of the one-step reaction and low cost of the maltose substrate. TreS has been purified from many other microorganisms and characterized [7][8][9][10][11] Pseudomonas stutzeri is a metabolically versatile saprophytic soil bacterium that exhibits extensive biotransformation potential and efficiently produces a wide range of bulk and fine chemicals. TreS was isolated from Pseudomonas stutzeri CJ38 and exogenously expressed in E. coli BL21 (DE3) [10].…”

“…TreS has been purified from many other microorganisms and characterized [7][8][9][10][11] Pseudomonas stutzeri is a metabolically versatile saprophytic soil bacterium that exhibits extensive biotransformation potential and efficiently produces a wide range of bulk and fine chemicals. TreS was isolated from Pseudomonas stutzeri CJ38 and exogenously expressed in E. coli BL21 (DE3) [10]. However, there is currently insufficient knowledge regarding the structural characteristics of this enzyme, including substrate specificity and transition state information.…”

Optimization of bioconversion process for trehalose production from enzymatic hydrolysis of kudzu root starch using a visualization method

“…Therefore, in the process of trehalose production, the degree of trehalose conversion rate (CR) was regarded as the most important property. The reaction condition parameters of the intramolecular transglycosylation from maltose into trehalose by TreS were summarized in many previous studies and listed in Table 4 (Jiang et al 2013;Ma et al 2006;Lee et al 2005;Xiuli et al 2009;Wang et al 2007b, c;Zhu et al 2008). Various combinations of pH (6-8.5), reaction temperature (15-40 °C), TreS concentration (10-40 %, 14,000 U/ml) and reaction time (18-36 h) are shown in Table 4 according to the orthogonal design scheme, together with the experimental results.…”

Clone and Expression of High Yield Recombinant Trehalose Synthase in Bacillus subtilis

“…But the main problem for trehalose synthase industrial production depends on its activity improvement. So far there are many reports about cloning and expression of trehalose synthase in E. coli [15][16][17][18]. However, E. coli is pathogenic and is not safe for food industry production.…”