Vidyasagar M, Prakash S B, Sreeramulu K
Department of Biochemistry, Gulbarga University, Gulbarga, 585106, India.
Lett Appl Microbiol. 2006 Oct;43(4):385-91. doi: 10.1111/j.1472-765X.2006.01980.x.
Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production.
Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions.
Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease.
The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.
分离和筛选产细胞外嗜盐嗜碱蛋白酶的极端嗜盐古菌，并优化培养条件以实现其最大产量。
从盐样品中分离出嗜盐几何菌属TSS101，并在含20%氯化钠的明胶和酪蛋白平板上筛选蛋白酶分泌情况。该古菌在装有50 ml（w/v）20%氯化钠、1%氯化镁、0.5%氯化钾、0.3%柠檬酸钠和1%蛋白胨、pH 7.2的250 ml烧瓶中，于40℃在旋转摇床上好氧培养。研究了在不同pH、温度、氯化钠浓度、金属离子以及不同碳源和氮源条件下的酶产量。部分纯化的蛋白酶在较宽的pH范围（7.0 - 10.0）内具有活性，在pH 10.0和温度60℃时活性最佳。该酶具有热稳定性，在80℃时保留70%的初始活性。在含有20%氯化钠（w/v）和1%脱脂奶粉的培养基中，于40℃振荡培养84 h后，蛋白酶产量最高。在7.0 - 10.0的较宽pH范围内观察到酶分泌。向培养基中添加200 mmol氯化钙可提高蛋白酶产量。富含蛋白质的面粉被证明是用于酶生产的廉价且良好的替代碳源。测试了不同渗透剂对嗜盐嗜碱蛋白酶生长和产量的影响，发现甜菜碱和甘油可促进生长但不分泌蛋白酶。固定化研究表明，固定在2%海藻酸钠珠中的全细胞在多达10批次的培养中保持稳定，并能够分泌蛋白酶，在振荡条件下60 h内可达到最大产量。
嗜盐几何菌属TSS101分泌一种细胞外嗜盐嗜碱且热稳定的蛋白酶。实现最大产量所需的最佳条件是20%氯化钠、1%脱脂奶粉和40℃的温度。添加200 mmol氯化钙可提高酶产量。在无氯化钠的情况下固定全细胞被证明可用于连续生产嗜盐嗜碱蛋白酶。
低成本的富含蛋白质的面粉被用作酶生产的替代碳源和氮源。将嗜盐细胞固定在海藻酸钠珠中可用于连续生产嗜盐酶。来自嗜盐几何菌属TSS101的嗜盐且热稳定的蛋白酶是工业应用的良好来源，并且可能是制备鱼露的合适来源。
