tailieunhanh - Expression and characterization of recombinant trehalose synthase in Bacillus subtilis

In this study, the treS gene was cloned from P. putida (VTCC 12263) and expressed in B. subtilis strain 1012 under the control of the promoter pgrac of pHT01 vector. This research provides the basis for production of safe recombinant TreS, which can be used in the food processing industry. | ACADEMIA JOURNAL OF BIOLOGY 2020 42 4 51 60 DOI 2615-9023 EXPRESSION AND CHARACTERIZATION OF RECOMBINANT TREHALOSE SYNTHASE IN Bacillus subtilis Nguyen Ngoc Trieu1 Nguyen Thi Quynh1 Nguyen Duc Hoang3 Nguyen Manh Dat4 Tran Duc Long1 2 Nguyen Thi Hong Loan1 2 1 Key Laboratory of Enzyme and Protein Technology VNUHN-University of Science 2 Faculty of Biology VNUHN-University of Science 3 Faculty of Biology and Biotechnology VNUHCM-University of Science 4 Food Industries Research Institute Ministry of Industry and Trade Received 17 April 2020 accepted 28 September 2020 ABSTRACT Trehalose synthase TreS EC 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 U g. Expression of TreS was highest when B. subtilis 1012 harboring pHT01-treS was cultured in TB medium at 30 oC induced with mM IPTG when OD600 reached and harvested after 10 hours of induction. The recombinant TreS purified by Ni-sepharose chromatography had specific activity of 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 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 . Keywords Bacillus subtilis cloning expression of recombinant protein enzyme characteristics trehalose synthase. Citation Nguyen Ngoc Trieu Nguyen Thi Quynh Nguyen Duc Hoang Nguyen Manh Dat Tran Duc Long Nguyen Thi .