Mn(2+) enhances theanine-forming activity of recombinant glutamine synthetase from Bacillus subtilis in Escherichia coli

• Published in: World journal of microbiology & biotechnology Vol. 24; no. 8; pp. 1267 - 1272
• Main Authors: Zhou, Xin, Zhang, Zhengping, Jia, Xiaohe, Wu, Yifan, Luo, Lan, Yin, Zhimin
• Format: Journal Article
• Language: English
• Published: 01.08.2008
• ISSN: 0959-3993
• DOI: 10.1007/s11274-007-9599-9

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4U/mg at a series concentration (0-100mM) of Mn(2+) at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in 'Materials and methods') and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2U/mg which is approximate to that (6.4U/mg) induced in LB in the presence of 10mM Mn(2+) at optimal pH7.5. The activity is markedly higher activated by Mn(2+) than that by other nine bivalent cations. Furthermore, M9-B (5kM Mn(2+) was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0ml reaction system with 0.1mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1g/L by paper chromatography and HPLC, respectively.