Department of Biochemistry, Sardar Bhagwan Singh PG Institute of Biomedical Sciences and Research, Balawala, Dehradun 248161, India.
Appl Biochem Biotechnol. 2012 Apr;166(7):1723-35. doi: 10.1007/s12010-012-9576-0. Epub 2012 Feb 26.
Purification and characterization of halotolerant, thermostable alkaline L-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH₄)₂SO₄ precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. L-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0-11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1-20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. L-Glutaminase is almost active with K⁺, Zn²⁺, and Ni²⁺ ions and K(m) and V(max) values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance, and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived L-glutaminase reported so far.
本文报道了从印度北阿坎德邦冈托里地区分离的芽孢杆菌 LKG-01（MTCC 10401）中分离得到的耐盐、耐热碱性 L-谷氨酰胺酶的纯化和特性。该酶通过（NH₄）₂SO₄沉淀、阴离子交换色谱和凝胶过滤，从无细胞提取物中得到 49 倍的纯化，回收率为 25%（比活 584.2 U/mg 蛋白）。酶的分子量为 66 kDa。L-谷氨酰胺酶在 pH 11.0 时最活跃，在 pH 8.0-11.0 范围内稳定。最适温度为 70°C，在 50°C 下预孵育 3 小时后完全稳定。该酶在 1-20%（w/v）NaCl 中显示出更高的活性，当 NaCl 浓度增加到 25%时，活性进一步降低到 80%。该酶对 K⁺、Zn²⁺和 Ni²⁺离子几乎具有活性，K(m)和 V(max)值分别为 240 μM 和 277.77 ± 1.1 U/mg 蛋白。与迄今为止报道的其他土壤微生物来源的 L-谷氨酰胺酶相比，该酶具有更高的比活、纯化倍数、更好的耐盐性和热稳定性，这使其更适合用于食品发酵。
