Purama Ravi Kiran, Goyal Arun
Department of Biotechnology, Indian Institute of Technology Guwahati, North Guwahati, Guwahati 781 039, Assam, India.
Bioresour Technol. 2008 Jun;99(9):3635-42. doi: 10.1016/j.biortech.2007.07.044. Epub 2007 Sep 24.
The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol fractionation (PEG) and gel-filtration. The cell free extract was subjected to fractionation by PEG-200, 400 and 1500. The 10% (w/v) PEG-1500 gave dextransucrase with maximum specific activity of 23 with 40 fold purification in a single step. The purified enzyme showed multiple molecular forms on SDS-PAGE, however the same sample showed a single band on non-denaturing native-PAGE. The purified dextransucrase fractions obtained from PEG-1500, confirmed the presence of dextran, when run on SDS-PAGE under non-denaturing gels for in situ activity detection by Periodic Acid Schiff's staining. The activity bands corresponded to the native and active form of the purified dextransucrase of approximately, 180kDa molecular size, that appeared on the denaturing gels stained with Coomassie Brilliant Blue. No bands appeared after staining the activity of dextransucrase on non denaturing SDS-PAGE gels with raffinose, which excluded the presence of fructosyltransferases. Further purification of 10% PEG-1500 purified dextransucrase by gel-filtration gave dextransucrase with specific activity of 35 with 61 fold purification.
采用聚乙二醇分级分离（PEG）和凝胶过滤法对肠系膜明串珠菌NRRL B - 640的胞外葡聚糖蔗糖酶进行了纯化。将无细胞提取物用PEG - 200、400和1500进行分级分离。10%（w/v）的PEG - 1500一步纯化得到的葡聚糖蔗糖酶比活性最高可达23，纯化倍数为40倍。纯化后的酶在SDS - PAGE上呈现多种分子形式，但在非变性天然PAGE上同一样品显示为单一条带。从PEG - 1500获得的纯化葡聚糖蔗糖酶级分，在非变性凝胶上进行SDS - PAGE并通过高碘酸希夫氏染色进行原位活性检测时，证实了葡聚糖的存在。活性条带对应于经考马斯亮蓝染色的变性凝胶上出现的分子量约为180kDa的纯化葡聚糖蔗糖酶的天然活性形式。用棉子糖对非变性SDS - PAGE凝胶上的葡聚糖蔗糖酶活性进行染色后未出现条带，这排除了果糖基转移酶的存在。通过凝胶过滤对10% PEG - 1500纯化的葡聚糖蔗糖酶进一步纯化，得到比活性为35、纯化倍数为61倍的葡聚糖蔗糖酶。
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