Clone and Expression of High Yield Recombinant Trehalose Synthase in Bacillus subtilis
Trehalose synthase is one kind of intermolecular transglucosylation enzyme, which catalyzes the conversion of maltose to trehalose. In this study the trehalose synthase gene was amplified from Pseudomonas putida P06 genomic DNA, ligated with pMA5 vector,
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Clone and Expression of High Yield Recombinant Trehalose Synthase in Bacillus subtilis Jing Su, Chunling Ma, Tengfei Wang, Piwu Li and Ruiming Wang
Abstract Trehalose synthase is one kind of intermolecular transglucosylation enzyme, which catalyzes the conversion of maltose to trehalose. In this study the trehalose synthase gene was amplified from Pseudomonas putida P06 genomic DNA, ligated with pMA5 vector, cloned into Bacillus subtilis WB800 and had good expression with the molecular weight of 77 KD. The recombinant trehalose synthase expression conditions were performed and enzyme reaction key parameters were investigated. The results showed the enzyme had optimal activity when it reacted for 2 h at 35 °C with pH value of 7.5 and the substrate concentration was 30 %. Trehalose content of samples was detected by HPLC and the enzyme activity reached to 318.12 U/ml in crude enzyme solution. This study is the first report about the expression of trehalose synthase in Bacillus subtilis, which lays the basis for trehalose large scale industrial production. Keywords Trehalose synthase Bacillus subtilis WB800
Pseudomonas putida
Gene expression
4.1 Introduction Trehalose is a non-reducing disaccharide, which has two glucose molecules linked in a, a-1, 1-glycosidic linkage. It widely spreads in bacteria, archaea, yeast, fungi, insects and a number of invertebrates [1]. It has high stability against extreme environment conditions such as temperature, pH and desiccation. So it plays important roles, such as a carbon energy reserve [2], a compatible solute under stress conditions [3–5] and a structural component of the cell wall. On the other J. Su C. Ma T. Wang P. Li R. Wang (&) School of Food and Bioengineering, Qilu University of Technology, Jinan 250300, China e-mail: [email protected]
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 249, DOI: 10.1007/978-3-642-37916-1_4, Ó Springer-Verlag Berlin Heidelberg 2014
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hand trehalose not only protects biomolecules in vivo but also has the same protection effect in vitro. This feature of trehalose has opened a new field for its application in food industry and pharmaceutical manufacturing practice [6, 7]. So far it is generally reported that there are at least three metabolized pathways for the biosynthesis of trehalose in microorganism. The first pathway is catalyzed by the trehalose-6-phosphate synthase, in which trehalose is synthesized through the glucosyl moiety from UDP-glucose to glucose-6-phosphate to form trehalose6-phosphate that further dephosphorylated to trehalose by trehalose-6-phosphate phosphatase [8, 9]. The second pathway involves the rearrangement of internal glycosidic linkage between the molecules of glucose polymer such as maltooligosaccharides produced from the hydrolysis of starch using a-amylase [10, 11], which can convert a-(1-4) linkage of the terminal residue of the maltooligosaccharides into a-(1–1) li
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