Centre for Advanced studies in Botany, University of Madras, Guindy Campus, Chennai, India.
J Basic Microbiol. 2011 Dec;51(6):614-24. doi: 10.1002/jobm.201000517. Epub 2011 Jul 21.
An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component.
一株分离自红藻 Kappaphycus alvarezii（Doty）Doty ex Silva 的巨大芽孢杆菌 RRM2 所产生的碱性丝氨酸蛋白酶，在本研究中首次进行了研究，并分析了其产生蛋白酶的情况。根据生化分析和 16S rDNA 序列分析对细菌进行了鉴定。从 B. megaterium RRM2 获得的胞外蛋白酶通过三步过程进行纯化，包括硫酸铵沉淀、凝胶过滤（Sephadex G100）和 Q-Sepharose 柱层析。纯度达到 30.6 倍，比活为 3591.5 U/mg 蛋白，分子量为 27 kDa。Ca(2+)、Mg(2+)、K(+)和 Na(+)等金属离子轻微增强了纯化酶的活性，而 Hg(2+)、Cu(2+)、Fe(2+)、CO(2+)和 Zn(2+)则降低了酶的活性。该酶在 pH 值为 9.0-10.0 的范围内具有活性，并且在 60°C 下仍保持活性。苯甲基磺酰氟（PMSF）抑制了酶的活性，因此证实该酶是一种碱性丝氨酸蛋白酶。同样，DTT 也抑制了酶的活性，从而证实了酶的二硫键性质。该酶对十二烷基硫酸钠（SDS）具有高度耐受性。当部分纯化的蛋白酶用作商业洗涤剂中的添加剂时，发现它是一种适合洗涤衣物的来源，特别是那些沾有血迹的衣物。此外，它在短时间内在山羊皮上显示出良好的脱毛活性，而不会影响其胶原蛋白成分。
