Mahapatra Samhita, Vickram A S, Sridharan T B, Parameswari R, Pathy M Ramesh
SBST, VIT University, Vellore, 632014, Tamil Nadu, India.
3 Biotech. 2016 Dec;6(2):213. doi: 10.1007/s13205-016-0530-7. Epub 2016 Oct 3.
An extracellular β-glucosidase was isolated from Proteus mirabilis VIT117 found to be growing on prawn shells. The enzyme production was found to be enhanced (14.58 U/ml) when the culture was maintained at pH 9 and provided with sorbitol as carbon source, yeast extract as nitrogen source and incubated at 37 °C for approximately 72 h. Statistical methods like Plackett-Burman and RSM were also applied here to study the effects of different combinations of growth parameters for the bacteria, where the most significant parameters were found to be inoculum size, pH, yeast extract, incubation time and sorbitol. The optimum concentrations of inoculum size, pH and yeast extract determined by RSM were 2 %, 9 and 2 %, respectively. Partial purification of the protein was done by ammonium sulfate precipitation, followed by dialysis, gel filtration chromatography and SDS-PAGE. The enzyme was found to have a molecular weight of approximately 50 kDa and was observed to be most active at 37 °C in pH 9, with a sharp decline in the enzyme activity when temperature or the pH was increased. Enzyme kinetics study was performed to understand the catalytic behavior of the enzyme and it was found that our β-glucosidase had 5.613 U/ml and 0.082 mM as V and K values, respectively.
从奇异变形杆菌VIT117中分离出一种胞外β-葡萄糖苷酶，该菌株能在虾壳上生长。研究发现，当培养物维持在pH 9，以山梨醇作为碳源、酵母提取物作为氮源，并在37℃下孵育约72小时时，酶产量会提高（14.58 U/ml）。这里还应用了Plackett-Burman和响应面法等统计方法来研究细菌生长参数不同组合的影响，其中最显著的参数是接种量、pH值、酵母提取物、孵育时间和山梨醇。通过响应面法确定的接种量、pH值和酵母提取物的最佳浓度分别为2%、9和2%。通过硫酸铵沉淀、透析、凝胶过滤色谱和SDS-PAGE对蛋白质进行了部分纯化。发现该酶的分子量约为50 kDa，在pH 9、37℃时活性最高，温度或pH值升高时酶活性急剧下降。进行了酶动力学研究以了解该酶的催化行为，发现我们的β-葡萄糖苷酶的Vmax和Km值分别为5.613 U/ml和0.082 mM。
